Heparinase III and uses thereof

ABSTRACT

The invention relates to heparinase III and mutants thereof. Modified forms of heparinase III having reduced enzymatic activity which are useful for a variety of purposes, including sequencing of heparin-like glycosaminoglycans (HLGAGs), removing active heparan sulfate from a solution, inhibition of angiogenesis, etc. have been discovered according to the invention. The invention in other aspects relates to methods of treating cancer and inhibiting tumor cell growth and/or metastasis using heparinase III, or products produced by enzymatic cleavage by heparinase III of HLGAGs.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/187,846 filed Mar. 8, 2000, the entire contents of which areincorporated herein by reference.

GOVERNMENT SUPPORT

Some aspects of the invention were made with government support underNIH Contract No. GM57073. The government may have certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to heparinase III and mutants thereof. Inparticular, the invention relates to modified forms of heparinase IIIhaving reduced enzymatic activity which are useful for a variety ofpurposes, including sequencing of heparin-like glycosaminoglycans(HLGAGs), removing HLGAGs from a solution, inhibition of angiogenesis,inhibiting coagulation, etc. The invention in other aspects relates tomethods of treating cancer and inhibiting tumor cell growth and/ormetastasis using heparinase III, or HLGAG products produced by enzymaticcleavage with heparinase III.

BACKGROUND OF THE INVENTION

Heparin like glycosaminoglycans (HLGAGs) are important components of theextracellular matrix that are believed to regulate a wide variety ofcellular activities including invasion, migration, proliferation andadhesion. Khodapkar, et al. 1998; Woods, et al., 1998) HLGAGs accomplishsome of these functions by binding to and regulating the biologicalactivities of diverse molecules, including growth factors, morphogens,enzymes, extracellular proteins. HLGAGs are linear polysaccharidescharacterized by a disaccharide-repeat unit of a uronic acid[α-L-iduronic acid (I) or β-D-glucuronic acid (G)] linked 1, 4 toα-B-hexosamine (H). (1) These polymers of 20-100 disaccharide units canbe additionally modified through N-and O-sulfation, epimerization at theC5 position of the uronic acid moiety, adding an additionalmicro-heterogenecity to these information dense molecules (1.5).

Although the structure and chemistry of HLGAGs are fairly wellunderstood, information on how specific HLGAG sequences modulatedifferent biological processes has proven harder to obtain. Theinventors have recently developed a rapid sequencing methodology forpolysaccharides using chemical and enzymatic tools to modify or degradean unknown HLGAG polymer in a sequence-specific manner. (Venkataraman,G., et al., Science, 286, 537-542 (1999), and U.S. patent applicationsSer. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000). Animportant enzymatic tool in this sequencing process is the heparinases,including heparinases I, II and III. The three heparinases are HLGAGdegrading enzymes which can be produced by Flavobacterium heparinum.Each of the heparinases has its own unique HLGAG sequence at which itcleaves, making these enzymes valuable tools in obtaining sequencespecific information. Heparinase I primarily cleaves HLGAGs at theH_(NS,6X)-I_(2S) ²-linkage found primarily in heparin-like regions(Ernst, S., et al., Crit, Rev. Biochem. Mol. Biol., 30, 387-444 (1995)).Desai, U., et al., Biochemistry, 32, 8140-8145 (1993)), and Jandik, K.,et al., Glycobiology, 4, 289-296 (1994)). Heparinase III cleaves at theH_(NAC)-I and H_(NY,6X)-G² linkages which are the major disaccharidesfound in heparan sulfate (Ernst, et al., (1995), supra, and Linhardt,R., et al., Biochemistry, 29, 2611-2617 (1990)). Heparinase II iscapable of recognizing and cleaving both sets of substrate linkages(Ernst, et al., (1995), supra). We have recently identified severalresidues which are critical to the activity of heparinase I andheparinase II. Cysteine 135 and histidine 203, as well as lysines 198,199, and 132 of heparinase I were found to be critical to the enzymaticactivity of the molecule. Cysteine 348 and histidines 238, 451, and 579were determined to be crucial for heparinase II activity. (Pending U.S.patent application Ser. No., 09/384,959; Sasisekharan, R., et al.,Biochemistry, 34, 14441-14448 (1995); Godavarti, R., et al.,Biochemistry, 35, 6846-6852 (1996); Godavarti, R., and Sasisekharan, R.,J. Biol. Chem. 273, 248-255 (1998); Shriver, Z., et al., J. Biol. Chem.,273, 22904-22912 (1998); and Shriver, Z., J. Biol. Chem., 273,10160-10167 (1998)).

Heparinase III is unique in that it is the only member of the heparinasefamily that recognizes and preferentially cleaves heparan sulfate.Heparinase III also contains no cysteines in its amino acid sequence.

Tumor metastasis involves the spread of tumor cells primarily via thevasculature to remote sites in the body. It is believed that as theextracellular matrix is degraded, the tumor cell-extracellular matrixinteractions are disassembled, freeing the tumor cell to extravagatethrough the capillary bed. Extraordinary progress has been made toelucidate the roles of collagen and related proteins, enzymes(collagenases and others) that degrade the extracellular matrix proteinsto regulate tumor angiogenesis and/or tumor cell invasion. It has alsorecently been hypothesized that HLGAG degrading enzymes, heparinases,assist in the breakdown of the extracellular matrix to regulate tumorgrowth, angiogenesis and metastasis. It has been suggested that theexpression of heparinases in association with tumor development,represents a switch from a metastatic tumor to a non-metastatic tumorand plays a role in initiating the process of metastasis. The hypothesiswas reaffirmed by recent cloning of a human heparinase gene and by thedemonstration of enhanced malignancy of cancer cells by over-expressionof the gene product for heparinase. (Hulett, et al., 1999 and Vlodavsky,et al., 1999).

SUMMARY OF THE INVENTION

It has been discovered, according to one aspect of the invention, thatexpression of heparinases does not necessarily represent a switch from aprimary tumor to a metastatic diseased state. Consistent with thecurrent paradigm, heparinase I activity was found to accelerate tumorgrowth and correlate with increased metastasis. Surprisingly, heparinaseIII, however, was found to inhibit primary tumor growth andsignificantly reduce metastasis. Thus, in one aspect the invention is amethod for preventing growth of a tumor by exposing a tumor cell to aneffective amount of heparinase III for preventing proliferation of thetumor cells in order to prevent growth of the tumor. In other aspects,the invention is a method for preventing tumor cell metastasis byexposing a tumor cell to an effective amount of heparinase III forpreventing invasion of the tumor cell across a barrier. The heparinaseIII may be a native heparinase III molecule or a modified heparinase IIImolecule. Native heparinase III may be synthesized or isolated.

Additionally, it has been discovered according to the invention thattherapeutic HLGAG fragments can be used to treat cancer. These fragmentsare useful for preventing the growth of a tumor as well as preventingmetastasis. These fragments can be generated by heparinase III treatmentof cancer cells. The fragments generated from the heparinase IIItreatment of a cancer cell can be used to prevent or treat cancer fromthe same or different cancer cells than are used to generate thefragments. Additionally, they can be used to treat or prevent cancer inthe same or a different subject than was used to generate the fragments.

The tumor cell can be exposed to the heparinase III by any method knownin the art. For instance, when the tumor cell is a tumor cell in vitro,heparinase III may be added to the in vitro culture. When the tumor cellis in vivo, the heparinase III may be administered by any method fordelivering the heparinase III to the tumor. For instance, in someembodiments the heparinase III may be administered systemically, such asby oral delivery, injection, etc. or locally, such as by directinjection into the tumor or tumor site or by direct application duringsurgical manipulation, etc.

The heparinase III may be administered alone or in conjunction withother therapies such as an anti-cancer drug. In some embodiments, thetumor is a prostate tumor or a melanoma.

In other aspects, the invention is a method for preparing therapeuticagents for the treatment of a tumor. The method involves isolating atleast a portion of a tumor, treating the portion of the tumor withheparinase III to produce HLGAG fragments, and isolating the HLGAGfragments, wherein the HLGAG fragments are the therapeutic agent. Insome embodiments, the method may also include the step of determiningthe sequence of the HLGAG fragments.

In other aspects of the invention, a method for treating a subjecthaving a tumor is provided. The method involves administering to thesubject therapeutic HLGAG fragments to treat the tumor. Optionally themethod may involve identifying a therapeutic HLGAG fragment byidentifying an HLGAG produced when the tumor is contacted withheparinase III. In some embodiments, the therapeutic HLGAG fragment is asynthetic HLGAG fragment generated based on the sequence of the HLGAGfragment identified when the tumor is contacted with heparinase III. Inother embodiments, the HLGAG fragment administered to the subject is anisolated HLGAG fragment produced when the tumor is contacted with theheparinase III.

In another aspect the invention is a method for treating or preventing asubject having a cancer or at risk of developing a cancer byadministering to the subject a therapeutic HLGAG fragment. In someembodiments the therapeutic HLGAG fragment is a composition of HLGAGfragments wherein at least 50%, 75%, or 90% of the HLGAG fragments aredi- or tri-sulfated disaccharides. In other embodiments the therapeuticHLGAG fragment is free of mono- or un-sulfated disaccharides.

According to another aspect of the invention, a composition is provided.The composition includes heparinase III or a therapeutic HLGAG fragmentin an effective amount for preventing metastasis of a tumor cell, and atargeting molecule for targeting the heparinase III to the tumor, in apharmaceutically-acceptable carrier. In some embodiments the heparinaseIII is a modified heparinase III and in other embodiments it is a nativeheparinase III. The targeting molecule may be, for instance, a compoundwhich binds specifically to an antigen on the surface of a tumor cell.

The invention in another aspect is a composition of a heparinase III ora therapeutic HLGAG fragment in an effective amount for preventingmetastasis of a tumor cell, and an anti-cancer compound in apharmaceutically-acceptable carrier.

The invention in other aspects is based on the identification ofimportant residues within the heparinase III molecule. It has beendiscovered according to the invention that modification of certainhistidine residues within the heparinase III molecule causes changes inthe enzymatic rate of the enzyme as well as the product profile producedby the enzyme. In particular, it was discovered that histidine 295 andhistidine 510 are important for enzymatic degradation of heparan sulfateby heparinase III. When these two histidines were changed to other aminoacids, all of the activity of the enzyme was lost. Modification of theother histidine residues resulted in changes in kinetic constants of theenzyme, but the enzyme still retained activity. Thus, in another aspectthe invention is a substantially pure heparinase III comprising apolypeptide having the amino acid sequence of the mature peptide of SEQID NO:2 or having conservative substitutions thereof within residuesnon-essential to enzymatic function, wherein at least one histidineresidue selected from the group consisting of His 36, His 105, His 110,His 139, His 152, His 225, His 234, His 241, His 424, His 469, and His539 has been substituted with a residue selected from the groupconsisting of alanine, serine, tyrosine, threonine, and lysine. In someembodiments the polypeptide has a substitution at His 110 or His 241. Inother aspects, the invention is a substantially pure heparinase IIIcomprising a polypeptide having the amino acid sequence of the maturepeptide of SEQ ID NO:2 or having conservative substitutions thereofwithin residues non-essential to enzymatic function, wherein at leastone histidine residue selected from the group consisting of His 295 andHis 510 has been substituted with any other amino acid.

In another aspect, the invention is a substantially pure heparinase IIIwhich is a modified heparinase III having a modified product profile,wherein the modified product profile of the modified heparinase III isat least 10% different than a native product profile of a nativeheparinase III.

In another aspect, the invention is a substantially pure heparinase IIIthat is a modified heparinase III that can cleave a HLGAG substratehaving a modified heparinase III k_(cat) value, wherein the modifiedheparinase III k_(cat) value is at least 10% different than a nativeheparinase III k_(cat) value. The invention also encompassespharmaceutical preparations of any of the substantially pure heparinaseIII molecules with a pharmaceutically-acceptable carrier. The inventionalso encompasses the modified heparinase III of the inventionimmobilized on a solid support membrane.

A method of specifically cleaving a HLGAG is provided according toanother aspect of the invention. The method of specifically cleaving aHLGAG includes the step of contacting an HLGAG with the modifiedheparinase III of the invention. In some embodiments, the method is amethod for preventing tumor cell proliferation or metastasis, asdescribed above. In other embodiments, the method is a method forsequencing HLGAGs. In yet other embodiments, the method is a method forremoving active HLGAGs from an HLGAG-containing fluid, a method forinhibiting angiogenesis, a method for inhibiting neovascularization,e.g., such as that associated with eye disease, a method for treatingpsoriasis, or a method for inhibiting coagulation.

The invention also includes a method for preparing LMWH by contacting anHLGAG sample with a modified heparinase III molecule to produce LMWH. Inother aspects the invention is a composition of the LMWH produced bythis method. In yet another aspect the invention is also a method fortreating or preventing a disorder associated with coagulation, tumor,psoriasis, or neovascularization, by administering to a subject aneffective amount of this composition to treat or prevent a disorderassociated with coagulation, tumor, psoriasis, or neovascularization.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the effect of DEPC inactivation ofheparinase III on rate constant.

FIG. 2 is a graph depicting the pH dependence of the second order rateconstant of inactivation upon incubation of heparinase III with varyingconcentrations of DEPC.

FIG. 3 is a graph depicting the quantification of DEPC-modifiedhistidine residues in heparinase III over a period of time.

FIG. 4 is a graph depicting the substrate protection of heparinase IIIinactivation by DEPC.

FIG. 5 is a reverse phase HPLC profile of a lys-C digest of heparinaseIII which was not exposed to DEPC (top panel) and a peptide profile ofheparinase III labeled with DEPC (bottom panel).

FIG. 6 is a series of graphs depicting SAX analysis of exhaustiveheparinase III digests of heparan sulfate. Heparan sulfate was digestedwith either heparinase III from F. heparinum (panel A), recombinantheparinase III (panel B), H295A mutant enzyme (panel C), H510A mutantenzyme (panel D), or the H105A mutant enzyme (panel E).

FIG. 7 depicts a circular dichroism analysis of recombinant heparinaseIII and the H295A mutant enzyme, and the H510A mutant enzyme.

FIG. 8, panel A, depicts the timecourse of the experiment on tumorgrowth and metastasis in tumor-bearing mice. FIG. 8, panel B, is a graphdepicting tumor volume in mice treated with PBS, inactive heparinaseIII, active heparinase III, and heparinase I.

FIG. 9 is a bar graph depicting number of lung nodules that developed 13days after tail vein injection of B16 BL6 cells. The cells were eithertreated with PBS, heparinase I, or heparinase III.

FIG. 10, panel A, depicts the tumor volume of mice that were treatedwith GAG fragments generated from treatment of B16 BL6 cells with eitherheparinase I, heparinase III, or PBS or fragments generated fromheparinase I treatment of LLC cells. Tumor volume was measured over timebetween 7 and 15 days post-injection of the tumor cells.

FIG. 10, panel B is a bar graph which quantitates the number of lungnodules of the mice described in panel A.

FIG. 11 is a bar graph depicting the effect on B16 cellular migrationand invasion of transfection with antisense 2OST in pcDNA3.1.

FIG. 12 shows bar graphs depicting the ability of the transfected cellsof FIG. 12 to develop into primary tumors as assessed by mean tumorvolume (12 a) and tumor weight (12 b).

FIG. 13, panel A, depicts the analysis of the heparinase 1-generatedfragments. FIG. 13, panel B, depicts the analysis of the heparinaseIII-generated fragments. FIG. 13, panel C, depicts the analysis with PBSas a control. FIG. 13, panel D, provides a table showing the relativepercentage of HLGAG disaccharides in the heparinase I and heparinaseIII-generated fragments. FIG. 13, panels E and F, show the massspectrometric oligosaccharide mapping of heparinase I and heparinase IIIderived HLGAG saccharide fragments.

FIG. 14 is a bar graph depicting FGF2 signaling modulated by HLGAGfragments

FIG. 15 is a table (15 a) and a schematic depicting the modulation ofFGF2 activity in vivo by B16BL6 fragments (15 b).

BRIEF DESCRIPTION OF THE SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence of heparinase III from F.bacterium.

Sequence ID No. 2 is the amino acid sequence of heparinase III from F.bacterium.

Sequence ID No. 3 is a peptide fragment.

DETAILED DESCRIPTION

The invention in some aspects relates to heparinase III, modified formsthereof and uses thereof. The invention arose from several scientificfindings which expand the field of heparinase biology. In particular theinvention is based in part on the discovery of new modified forms ofheparinase that have varying enzymatic activity and produce differingproduct profiles. The invention is also based on the finding that nativeheparinase III, modified forms of heparinase III, and modified forms ofheparinase II having heparinase III like activity are useful for thetreatment and prevention of tumor cell growth and metastasis.

The present invention provides a series of new modified heparinase IIImolecules. In particular, based upon a detailed structural andfunctional characterization of heparinase III, new heparinases withaltered stability, activity and specificity are provided. The modifiedheparinases of the invention have many in vivo, in vitro and ex vivoutilities. For instance, they have great value in generating lowmolecular weight HLGAGs, heparan sulfate, or heparan sulfate fragmentsfor clinical use. Additionally they can be used to neutralize thefunction of heparan sulfate containing HLGAGs or they can be used toidentify the sequence of HLGAGs. Other uses are described herein.

Heparinase III is unique in that it is the only member of the heparinasefamily that recognizes and cleaves heparan sulfate as its onlysubstrate. Heparinase III is also unique among its heparin-degradingfamily members in that it contains no cysteines in its primary aminoacid sequence (Su, H., Blain, F., Musil, R. A., Zimmermann, J. J., Gu,K., and Bennett, D. C. (1996) Appl. Environ. Micro. 62,2723-34 andGodavarti, R., Davis, M., Venkataraman, G., Cooney, C. L., Langer, R.,and Sasisekharan, R. (1996) Biochem. and Biophys. Res. Comm. 225,751-58). Heparinase III, however, does contain thirteen histidines ofwhich one or several might be involved in the activity of the enzyme.Through a combination of chemical modification, peptide mapping, andsite-directed mutagenesis studies, the role of histidines in thecatalytic activity of heparinase III has been identified, according tothe invention.

The nucleotide and amino acid sequences of heparinase III are providedin SEQ ID NO: 1 and SEQ ID NO: 2. The sequence of heparinase III hasbeen reported in Su, H., Blain, F., Musil, R. A., Zimmermann, J. J., Gu,K., and Bennett, D. C. (1996) Appl. Environ. Micro. 62, 2723-34. andGodavarti, R., Davis, M., Venkataraman, G., Cooney, C. L., Langer, R.,and Sasisekharan, R. (1996) Biochem. and Biophys. Res. Comm. 225,751-58, U.S. Pat. Nos. 5,919,693 and 5,681,733, and is listed inAccession number 171365. These sequences have provided the first insightinto the primary structure of the native heparinase III of F. heparinum.

The present disclosure provides additional information about thesecondary and tertiary structure of the heparinase III, as well as,information relating to the functional roles of the various regions ofthe enzyme. This information is based upon detailed biochemical mappingof the important sites within the enzyme and characterization of thesesites through kinetic studies, characterization of mutants created bysite-directed mutagenesis, etc. The result is a detailed picture of theprimary, secondary, and tertiary structures of heparinase III and thefunctional roles of various regions of the enzyme as well as thefunctions of specific mutants thereof.

The invention is based on several scientific findings. It was discoveredaccording to the invention that various amino acid residues withinheparinase III are essential to the catalytic function of these enzymesand can be modified to alter the enzymatic activity of these compounds.It was also discovered that other amino acid residues are absolutelycritical to the function of heparinase III and if they are substitutedor modified the activity of these compounds is lost completely. Inparticular, it has been shown according to the invention through acombination of chemical modification, peptide mapping, and site-directedmutagenesis experiments that two histidines, histidine 295 and histidine510, are critical for the enzymatic degradation of HLGAGs by heparinase

As shown in the Examples section, DEPC was used in the first step of theanalysis of heparinase III. DEPC is extremely useful in elucidating therole of histidines in enzymatic function. Care has to be taken, though,to ensure that DEPC doesn't modify other nucleophilic amino acids suchas tyrosine, lysine or cysteine (Godavarti, R., Cooney, C. L., Langer,R., and Sasisekharan, R. (1996) Biochemistry 35, 6846-52 and Shriver,Z., Hu, Y., and Sasisekharan, R. (1998) J. Biol. Chem. 273, 10160-67).In the case of heparinase III, there are no cysteine residues in theprimary amino acid sequence, eliminating this amino acid as a potentialconfounding factor in the chemical modification studies. Also, nodecrease in the absorbance at 278 nm was observed after heparinase IIIwas incubated with DEPC, indicating that tyrosine residues were notmodified. An increase in the inactivation kinetics without a change inthe order of the reaction was observed from pH 6.0-7.5 upon DEPCtreatment. Furthermore, the DEPC modification was 90% reversible uponincubation with 300 mM hydroxylamine. Above pH 8.0, the inactivationkinetics were no longer first order for DEPC and the modification couldnot be reversed by hydroxylamine, indicating that residues other thanhistidines (i.e. lysines) were being modified at those pHs. However, atneutral pH, the data indicates that DEPC specifically modifies thehistidine residues of heparinase III.

Consistent with the observation that DEPC is modifying a histidineresidue, there was an increase in the absorbance at 240 nm as a functionof time. This is indicative of formation of an N-carbethoxyhistidylderivative, the product of a reaction between DEPC and a histidineresidue. Over the course of ten minutes, 1.8 histidine residues weremodified and the enzymatic activity was decreased by 90%. Also,pre-incubation with heparan sulfate resulted in lower inactivationkinetics of heparinase III by DEPC. These data indicated that DEPCspecifically modified a critical histidine residue proximate to thesubstrate binding/active site of heparinase III, inactivating theenzyme.

An apparent discrepancy arose from these results in that the reaction ofDEPC with heparinase III follows pseudo-first order kinetics, yet twohistidines appeared to be independently modified, suggesting that twosurface accessible histidines react with DEPC at identical rates. Itcould be the case that either one or both of the modified residues isresponsible for inactivating the enzyme. Site-directed mutagenesisexperiments were performed to determine if two histidines were essentialfor heparinase III's catalytic activity. The results from thesite-directed mutagenesis experiments confirmed and expanded upon thechemical modification data in that surface accessible histidines arecritical for heparinase III activity. These results identify histidine295 and histidine 510 as the primary histidines involved in thedegradation of HLGAGs by heparinase III. When these residues arereplaced with alanines, the enzyme loses all activity towards itssubstrate. None of the other histidine residues when mutated to alanineshow a complete loss of activity. The results from the peptide mappingstudies confirm the importance of the surface accessibility of histidine295.

The loss of activity with the H295A and H510A enzymes can be explainedin several ways. It may be that these histidines are necessary forproper folding of heparinase III. However, the CD spectrum of H295A,H510A, and recombinant heparinase III were nearly identical, stronglyindicating that this is not the case. It is more likely that histidine295 and histidine 510 play a direct role in the binding of HLGAGs to theenzyme or that histidine 295 and histidine 510 are critical active siteresidues directly involved in the catalytic degradation of HLGAGs.Modified heparinase III molecules having a change in amino acid at His295 or 510 can be useful for a variety of purposes, e.g., as acompetitive inhibitor to functional heparinase III.

The studies described in the Examples section also identified severalheparinase III mutants which had altered levels of activity but whichwere still active. These mutants include heparinase III molecules havingthe following residues mutated or substituted: His36, His105, His110,His139, His152, His225, His234, His241, His424, His469, and His539.Thus, the present invention provides for novel modified heparinasesrationally designed on the basis of the sequence of the heparinase IIIof F. heparinum and the structural and functional characterizationsdisclosed herein.

In the description herein, reference is made to the amino acid residuesand residue positions of native heparinase III disclosed in SEQ ID NO 2.In particular, residues and residue positions are referred to as“corresponding to” a particular residue or residue position ofheparinase III. As will be obvious to one of ordinary skill in the art,these positions are relative and, therefore, insertions or deletions ofone or more residues would have the effect of altering the numbering ofdownstream residues. In particular, N-terminal insertions or deletionswould alter the numbering of all subsequent residues. Therefore, as usedherein, a residue in a recombinant modified heparinase will be referredto as “corresponding to” a residue of the full heparinase III if, usingstandard sequence comparison programs, they would be aligned. Many suchsequence alignment programs are now available to one of ordinary skillin the art and their use in sequence comparisons has become standard. Asused herein, this convention of referring to the positions of residuesof the recombinant modified heparinases by their correspondingheparinase III residues shall extend not only to embodiments includingN-terminal insertions or deletions but also to internal insertions ordeletions (e.g., insertions or deletions in “loop” regions).

In addition, in the description herein, certain substitutions of oneamino acid residue for another in a recombinant modified heparinase arereferred to as “conservative substitutions.” As used herein, a“conservative amino acid substitution” or “conservative substitution”refers to an amino acid substitution in which the substituted amino acidresidue is of similar charge as the replaced residue and is of similaror smaller size than the replaced residue. Conservative substitutions ofamino acids include substitutions made amongst amino acids within thefollowing groups: (a) the small non-polar amino acids, A, M, I, L, andV; (b) the small polar amino acids, G, S, T and C; (c) the amido aminoacids, Q and N; (d) the aromatic amino acids, F, Y and W; (e) the basicamino acids, K, R and H; and (f) the acidic amino acids, E and D.Substitutions which are charge neutral and which replace a residue witha smaller residue may also be considered “conservative substitutions”even if the residues are in different groups (e.g., replacement ofphenylalanine with the smaller isoleucine). The term “conservative aminoacid substitution” also refers to the use of amino acid analogs orvariants.

Methods for making amino acid substitutions, additions or deletions arewell known in the art and are described in detail in the Examples below.The terms “conservative substitution”, “non-conservative substitutions”,“non-polar amino acids”, “polar amino acids”, and “acidic amino acids”are all used consistently with the prior art terminology. Each of theseterms is well-known in the art and has been extensively described innumerous publications, including standard biochemistry text books, suchas “Biochemistry” by Geoffrey Zubay, Addison-Wesley Publishing Co., 1986edition, which describes conservative and non-conservativesubstitutions, and properties of amino acids which lead to theirdefinition as polar, non-polar or acidic.

Even when it is difficult to predict the exact effect of a substitutionin advance of doing so, one skilled in the art will appreciate that theeffect can be evaluated by routine screening assays, preferably thebiological assays described herein. Modifications of peptide propertiesincluding thermal stability, hydrophobicity, susceptibility toproteolytic degradation or the tendency to aggregate with carriers orinto multimers are assayed by methods well known to the ordinarilyskilled artisan. For additional detailed description of proteinchemistry and structure, see Schulz, G. E. et al., Principles of ProteinStructure, Springer-Verlag, New York, 1979, and Creighton, T. E.,Proteins: Structure and Molecular Principles, W. H. Freeman & Co., SanFrancisco, 1984.

Additionally, some of the amino acid substitutions are non-conservativesubstitutions. In certain embodiments where the substitution is remotefrom the active or binding sites, the non-conservative substitutions areeasily tolerated provided that they preserve the tertiary structurecharacteristic of native heparinase, thereby preserving the active andbinding sites. Non-conservative substitutions, such as between, ratherthan within, the above groups (or two other amino acid groups not shownabove), which will differ more significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

In one aspect, the invention is a substantially pure heparinase which isa modified heparinase III having a modified heparinase III k_(cat)value, wherein the modified heparinase III k_(cat) value is at least 10%different than a native heparinase III k_(cat) value. In a preferredembodiment, the modified heparinase III k_(cat) value is at least 20%different than a native heparinase III k_(cat) value. In anotherpreferred embodiment the modified heparinase III k_(cat) value is atleast 50% different than a native heparinase III k_(cat) value. A“modified heparinase III k_(cat) value” as used herein is a measurementof the catalytic activity of the modified heparinase III enzyme withrespect to a heparan sulfate-like glycosaminoglycan substrate.

The k_(cat) value may be determined using any enzymatic activity assaywhich is useful for assessing the activity of a heparinase enzyme, suchas the assays set forth in the Examples below. Several such assays arewell-known in the art. For instance, an assay for measuring k_(cat) isdescribed in (Ernst, S. E., Venkataraman, G., Winkler, S., Godavarti,R., Langer, R., Cooney, C. and Sasisekharan. R. (1996) Biochem. J. 315,589-597. The “native heparinase III k_(cat) value” is the measure ofenzymatic activity of the native heparinase III.

The modified heparinase may have a reduced enzymatic activity withrespect to HLGAGs. A “reduced enzymatic activity” is assessed bycomparing the k_(cat) value of the modified heparinase with that ofnative heparinase. Preferably the k_(cat) value of the modifiedheparinase III will be less than or equal to 75% of the nativeheparinase III k_(cat) value. A modified heparinase having reducedenzymatic activity with respect to HLGAGs is one which has modificationsin the residues essential for catalytic activity. For instance, mutationof His¹¹⁰ or His²⁴¹ causes the heparinase III to have a reducedenzymatic activity. A modified heparinase III which has a increasedenzymatic activity is one which has altered residues which produce anenzyme with greater enzymatic activity. For instance, mutation of His¹³⁹produces modified heparinase III molecules having increased enzymaticactivity. Additionally, when His²²⁵ is mutated in heparinase III, amodified heparinase III is produced which displays nearly the sameenzymatic activity as native heparinase III. These enzymes are alsouseful.

As used herein, with respect to heparinases, the term “substantiallypure” means that the heparinases are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use. Inparticular, the heparinases are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,producing pharmaceutical preparations or sequencing. Because theheparinases of the invention may be admixed with a pharmaceuticallyacceptable carrier in a pharmaceutical preparation, the heparinase maycomprise only a small percentage by weight of the preparation. Theheparinase is nonetheless substantially pure in that it has beensubstantially separated from the substances with which it may beassociated in living systems.

Based on the disclosure provided herein, those of ordinary skill in theart will be able to identify other modified heparinase III moleculeshaving altered enzymatic activity with respect to the native heparinaseIII molecule.

In another aspect, the invention is a substantially pure heparinasewhich is a modified heparinase III having a modified product profile,wherein the modified product profile of the modified heparinase III isat least 10% different than a native product profile of a nativeheparinase III. Preferably it is at least 20% or even at least 50%. A“modified product profile” as used herein is a set of degradationproducts produced by a modified heparinase which differ from thedegradation products which are produced by a native heparinase underidentical enzymatic conditions. The difference in the product profilemay be due to the presence of different enzymatic products or simply inthe number of enzymatic products formed by the modified heparinasecompared to the native heparinase, or a combination of the two. Forinstance, the formation of different enzymatic products by a modifiedheparinase as opposed to the native heparinase, would constitute amodified product profile. Additionally, the production of the same typesof enzymatic products but in a lesser or greater amount by the modifiedheparinase as opposed to the native heparinase, would also constitute amodified product profile.

The product profile produced by a modified heparinase or a nativeheparinase may be determined by any method known in the art forexamining the type or quantity of degradation product produced byheparinase. One preferred method for determining the type and quantityof product is described in Rhomberg, A. J. et al., PNAS, v. 95, p.4176-4181 (April 1998), which is hereby incorporated in its entirety byreference. The method disclosed in the Rhomberg reference utilizes acombination of mass spectrometry and capillary electrophoretictechniques to identify the enzymatic products produced by heparinase.The Rhomberg study utilizes heparinase to degrade HLGAGs to produceHLGAG oligosaccharides. MALDI (Matrix-Assisted Laser DesorptionIonization) mass spectrometry can be used for the identification andsemiquantitative measurement of substrates, enzymes, and end products inthe enzymatic reaction. The capillary electrophoresis techniqueseparates the products to resolve even small differences amongst theproducts and is applied in combination with mass spectrometry toquantitate the products produced. Capillary electrophoresis may evenresolve the difference between a disaccharide and its semicarbazonederivative. Detailed methods for sequencing polysaccharides and otherpolymers are disclosed in co-pending U.S. patent applications Ser. Nos.09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and having commoninventorship. The entire contents of both applications are herebyincorporated by reference.

Briefly, the method is performed by enzymatic digestion, followed bymass spectrometry and capillary electrophoresis. The enzymatic assayscan be performed in a variety of manners, as long as the assays areperformed identically on the modified heparinase and the nativeheparinase, so that the results may be compared. In the exampledescribed in the Rhomberg reference, enzymatic reactions are performedby adding 1 mL of enzyme solution to 5 mL of substrate solution. Thedigestion is then carried out at room temperature (22° C.), and thereaction is stopped at various time points by removing 0.5 mL of thereaction mixture and adding it to 4.5 mL of a MALDI matrix solution,such as caffeic acid (approximately 12 mg/mL) and 70%acetonitrile/water. The reaction mixture is then subjected to MALDI massspectrometry. The MALDI surface is prepared by the method of Xiang andBeavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8,199-204). A two-fold lower access of basic peptide (Arg/Gly)₁₅ ispremixed with matrix before being added to the oligosaccharide solution.A 1 mL aliquot of sample/matrix mixture containing 1-3 picomoles ofoligosaccharide is deposited on the surface. After crystallizationoccurs (typically within 60 seconds), excess liquid is rinsed off withwater. MALDI mass spectrometry spectra is then acquired in the linearmode by using a PerSeptive Biosystems (Framingham, Mass.) Voyager Elitereflectron time-of-flight instrument fitted with a 337 nanometernitrogen laser. Delayed extraction is used to increase resolution (22kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gateat 1,000, 128 shots averaged). Mass spectra are calibrated externally byusing the signals for proteinated (Arg/Gly)₁₅ and its complex with theoligosaccharide.

Capillary electrophoresis is then performed on a Hewlett-Packard^(3D) CEunit by using uncoated fused silica capillaries (internal diameter 75micrometers, outer diameter 363 micrometers, 1_(det) 72.1 cm, and1_(tot) 85 cm). Analytes are monitored by using UV detection at 230 mnand an extended light path cell (Hewlett-Packard). The electrolyte is asolution of 10 mL dextran sulfate and 50 millimolar Tris/phosphoric acid(pH2.5). Dextran sulfate is used to suppress nonspecific interactions ofthe heparin oligosaccharides with a silica wall. Separations are carriedout at 30 kV with the anode at the detector side (reversed polarity). Amixture of a ⅕-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid(10 micromolar each) is used as an internal standard.

Other methods for assessing the product profile may also be utilized.For instance, other methods include methods which rely on parameterssuch as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994),Glycobiology, 4:284-296) or total UV absorbance (Ernst, S. et al.,(1996), Biochem. J., 315:589-597) or mass spectrometry or capillaryelectrophoresis alone.

The modified heparinases of the invention may be used for any of thesame purposes as native heparinase III. For instance, the modifiedheparinase III molecules can be used to specifically cleave a HLGAG bycontacting the HLGAG substrate with one of the modified heparinases ofthe invention. The invention is useful in a variety of in vitro, in vivoand ex vivo methods in which it is useful to cleave HLGAGs.

The modified heparinase III may be used, for instance, in a method forinhibiting angiogenesis. In this method an effective amount forinhibiting angiogenesis of the heparinase III is administered to asubject in need of treatment thereof. Angiogenesis as used herein is theinappropriate formation of new blood vessels. “Angiogenesis” oftenoccurs in tumors when endothelial cells secrete a group of growthfactors that are mitogenic for endothelium causing the elongation andproliferation of endothelial cells which results in a generation of newblood vessels. Several of the angiogenic mitogens are heparin or heparansulfate binding peptides which are related to endothelial cell growthfactors.

The modified heparinases are also useful for treating or preventingcancer cell growth or metastasis. This aspect of the invention isdiscussed in more detail below, with respect to both native and modifiedheparinase III.

The modified heparinases are also useful for inhibitingneovascularization associated with disease such as eye disease.Neovascularization, or angiogenesis, is the growth and development ofnew arteries. It is critical to the normal development of the vascularsystem, including injury-repair. There are, however, conditionscharacterized by abnormal neovascularization, including diabeticretinopathy, neovascular glaucoma, rheumatoid arthritis, and certaincancers. For example, diabetic retinopathy is a leading cause ofblindness. There are two types of diabetic retinopathy, simple andproliferative. Proliferative retinopathy is characterized byneovascularization and scarring. About one-half of those patients withproliferative retinopathy progress to blindness within about five years.

Another example of abnormal neovascularization is that associated withsolid tumors. It is now established that unrestricted growth of tumorsis dependant upon angiogenesis, and that induction of angiogenesis byliberation of angiogenic factors can be an important step incarcinogenesis. For example, basic fibroblast growth factor (bFGF) isliberated by several cancer cells and plays a crucial role in cancerangiogenesis. As used herein, an angiogenic condition means a disease orundesirable medical condition having a pathology includingneovascularization. Such diseases or conditions include diabeticretinopathy, neovascular glaucoma and rheumatoid arthritis (non-cancerangiogenic conditions). Cancer angiogenic conditions are solid tumorsand cancers or tumors otherwise associated with neovascularization suchas hemangioendotheliomas, hemangiomas and Kaposi's sarcoma.

Proliferation of endothelial and vascular smooth muscle cells is themain feature of neovascularization. Thus the modified heparinase III ofthe invention is useful for preventing proliferation and, therefore,inhibiting or arresting altogether the progression of the angiogeniccondition which depends in whole or in part upon suchneovascularization.

Neovascularization and angiogenesis are also important in a number ofother pathological processes, including arthritis, psoriasis, diabeticretinopathy, chronic inflammation, scleroderma, hemangioma, retrolentalfibroplasia and abnormal capillary proliferation in hemophiliac joints,prolonged menstruation and bleeding, and other disorders of the femalereproductive system (J. Folkman, Nature Medicine, Vol 1, p. 27-31,(1995); J. W. Miller, et al., J. Pathol., Vol. 145, pp. 574-584 (1994);A. P. Adamid, et al., Amer. J. Ophthal., Vol. 118, pp. 445-450 (1994);K. Takahashi, at al., J. Clin. Invest., Vol. 93, pp. 2357-2364 (1994);D. J. Peacock, et al., J. Exp. Med., Vol. 175, pp. 1135-1138 (1992); B.J. Nickoloff, et al., Amer. J. Pathol., Vol. 44, pp. 820-828 (1994); J.Folkman, Steroid Hormones and Uterine Bleeding, N. J. Alexander and C.d'Arcangues, Eds., American Association for the Advancement of SciencePress, Washington, D.C., U.S.A., pp. 144-158 (1992)). Thus, in anotherembodiment, the modified heparinase is administered to treat diseasessuch as psoriasis. Psoriasis is a common dermatological disease causedby chronic inflammation.

The H295A and H510A modified heparinases are also useful according tothe invention as inhibitors of heparinase III activity. These modifiedheparinases have a minimum one base pair modification from nativeheparinase but have no enzymatic activity. Thus, modified heparinaseshaving a H295A or H510A modification can be used as competitiveinhibitors of native or functional modified forms of heparinase III.These compounds are useful any time it is desirable to block heparinaseIII activity, e.g., when cell proliferation and migration is desirableor to block the activity of heparinase III in a solution.

The modified heparinases of the invention are also useful as tools forsequencing HLGAGs. Detailed methods for sequencing polysaccharides andother polymers are disclosed in co-pending U.S. patent applications Ser.Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and havingcommon inventorship. These methods utilize tools such as heparinases inthe sequencing process. The modified heparinase III of the invention isuseful as such a tool.

The modified heparinases of the invention may also be used to removeactive HLGAGs from a HLGAG containing fluid. A HLGAG containing fluid iscontacted with the modified heparinase III of the invention to degradethe HLGAG. The method is particularly useful for the ex vivo removal ofHLGAGs from blood. In one embodiment of the invention the modifiedheparinase is immobilized on a solid support as is conventional in theart. The solid support containing the immobilized modified heparinasemay be used in extracorporeal medical devices (e.g. hemodialyzer,pump-oxygenator) for systemic heparinization to prevent the blood in thedevice from clotting. The support membrane containing immobilizedheparinase III is positioned at the end of the device to neutralize theHLGAG before the blood is returned to the body.

In another aspect, the invention is an immobilized substantially pureheparinase of the invention. The heparinase may be immobilized to anytype of support but if the support is to be used in vivo or ex vivo itis desired that the support is sterile and biocompatible. Abiocompatible support is one which would not cause an immune or othertype of damaging reaction when used in a subject. The heparinase may beimmobilized by any method known in the art. Many methods are known forimmobilizing proteins to supports.

The heparinase III is, in some embodiments, immobilized on a solidsupport. A “solid support” as used herein refers to any solid materialto which a protein can be immobilized. Solid supports, for example,include but are not limited to membranes, e.g., natural and modifiedcelluloses such as nitrocellulose or nylon, Sepharose, Agarose, glass,polystyrene, polypropylene, polyethylene, dextran, amylases,polyacrylamides, polyvinylidene difluoride, other agaroses, andmagnetite, including magnetic beads. The carrier can be totallyinsoluble or partially soluble and may have any possible structuralconfiguration. Thus, the support may be spherical, as in a bead, orcylindrical, as in the inside surface of a test tube or microplate well,or the external surface of a rod. Alternatively, the surface may be flatsuch as a sheet, test strip, bottom surface of a microplate well, etc.

The modified heparinase III molecules are also useful for generatingLMWHs which have many therapeutic utilities. The modified heparinase IIImolecules and LMWH can be used for the treatment of any type ofcondition in which LMWH therapy has been identified as a useful therapy,e.g., preventing coagulation, preventing psoriasis.

Thus, the modified heparinase molecules are useful for treating orpreventing disorders associated with coagulation. A “disease associatedwith coagulation” as used herein refers to a condition characterized bylocal inflammation resulting from an interruption in the blood supply toa tissue due to a blockage of the blood vessel responsible for supplyingblood to the tissue such as is seen for myocardial or cerebralinfarction. A cerebral ischemic attack or cerebral ischemia is a form ofischemic condition in which the blood supply to the brain is blocked.This interruption in the blood supply to the brain may result from avariety of causes, including an intrinsic blockage or occlusion of theblood vessel itself, a remotely originated source of occlusion,decreased perfusion pressure or increased blood viscosity resulting ininadequate cerebral blood flow, or a ruptured blood vessel in thesubarachnoid space or intracerebral tissue.

The methods of the invention are useful also for treating cerebralischemia. Cerebral ischemia may result in either transient or permanentdeficits and the seriousness of the neurological damage in a patient whohas experienced cerebral ischemia depends on the intensity and durationof the ischemic event. A transient ischemic attack is one in which theblood flow to the brain is interrupted only briefly and causes temporaryneurological deficits, which often are clear in less than 24 hours.Symptoms of TIA include numbness or weakness of face or limbs, loss ofthe ability to speak clearly and/or to understand the speech of others,a loss of vision or dimness of vision, and a feeling of dizziness.Permanent cerebral ischemic attacks, also called stroke, are caused by alonger interruption in blood flow to the brain resulting from either athromboembolism. A stroke causes a loss of neurons typically resultingin a neurologic deficit that may improve but that does not entirelyresolve. Thromboembolic stroke is due to the occlusion of anextracranial or intracranial blood vessel by a thrombus or embolus.Because it is often difficult to discern whether a stroke is caused by athrombosis or an embolism, the term “thromboembolism” is used to coverstrokes caused by either of these mechanisms.

The methods of the invention in some embodiments are directed to thetreatment of acute thromboembolic stroke using modified heparinase IIIor the LMWHs generated therewith. An acute stroke is a medical syndromeinvolving neurological injury resulting from an ischemic event, which isan interruption in the blood supply to the brain.

An effective amount of a modified heparinase III or the LMWHs generatedtherewith alone or in combination with another therapeutic for thetreatment of stroke is that amount sufficient to reduce in vivo braininjury resulting from the stroke. A reduction of brain injury is anyprevention of injury to the brain which otherwise would have occurred ina subject experiencing a thromboembolic stroke absent the treatment ofthe invention. Several physiological parameters may be used to assessreduction of brain injury, including smaller infarct size, improvedregional cerebral blood flow, and decreased intracranial pressure, forexample, as compared to pretreatment patient parameters, untreatedstroke patients or stroke patients treated with thrombolytic agentsalone.

The modified heparinase III or the LMWHs generated therewith may be usedalone or in combination with a therapeutic agent for treating a diseaseassociated with coagulation. Examples of therapeutics useful in thetreatment of diseases associated with coagulation includeanticoagulation agents, antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Antiplatelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics and also antithrombin agents such as, but not limited to,hirudin.

Thrombolytic agents lyse clots which cause the thromboembolic stroke.Thrombolytic agents have been used in the treatment of acute venousthromboembolism and pulmonary emboli and are well known in the art (e.g.see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S(1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p.10S-17S(1995)). Thrombolytic agents include, but are not limited to,plasminogen, a₂-antiplasmin, streptokinase, antistreplase, tissueplasminogen activator (tPA), and urokinase. “tPA” as used hereinincludes native tPA and recombinant tPA, as well as modified forms oftPA that retain the enzymatic or fibrinolytic activities of native tPA.The enzymatic activity of tPA can be measured by assessing the abilityof the molecule to convert plasminogen to plasmin. The fibrinolyticactivity of tPA may be determined by any in vitro clot lysis activityknown in the art, such as the purified clot lysis assay described byCarlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modifiedform described by Bennett, W. F. Et al., 1991, Supra, the entirecontents of which are hereby incorporated by reference.

The invention also relates to the discovery that heparinase III,modified forms thereof, modified forms of heparinase II and degradationproducts of heparinases (HLGAG fragments) actually are useful fortreating and preventing cancer cell proliferation and metastasis. Thus,according to another aspect of the invention, there is provided methodsfor treating subjects having or at risk of having cancer.

Heparinases degrade HLGAGs, which are linear polysaccharidescharacterized by a disaccharide-repeat unit of a uronic acid[α-L-iduronic acid (I) or β-D-glucuronic acid (G)] linked 1,4 toα-D-hexosamine (H). HLGAGs are the most acidic, heterogeneous andinformation dense biopolymer found in nature due to the highly variablechemical modification of the disaccharide repeat unit—primarily in theform of sulfation at the N-, 3O and 6O positions of H, and the 2O of theuronic acids. Critically, HLGAGs (along with collagen) are keycomponents of the cell surface-extracellular matrix (ECM) interface.While collagen-like proteins provide the necessary extracellularscaffold for cells to attach and form tissues, the complexpolysaccharides fill the space created by the scaffold and act as amolecular sponge by specifically binding and regulating the biologicalactivities of numerous signaling molecules like growth factors,cytokines etc. It has recently been recognized that cells synthesizedistinct HLGAG sequences and decorate themselves with these sequences,using the extraordinary information content present in the sequences tobind specifically to many signaling molecules and thereby regulatevarious biological processes.

Tumor metastasis involves the spread of tumor cells primarily via thevasculature following the disassembly of tumor cell-ECM interactionsthrough the degradation of the ECM, and tumor cell extravasation throughthe capillary bed. Recent evidence has suggested that collagen (andrelated proteins), enzymes (collagenases and others) that degrade theproteinaceous component of the ECM may play roles in the regulation oftumor angiogenesis or tumor cell invasion of the ECM. However, thechemical heterogeneity of complex polysaccharides and lack of effectivetools, has seriously limited investigations into the roles of HLGAGs intumor growth and metastasis. Interestingly, however, in parallel withcollagen and the proteases, it has been hypothesized that HLGAGdegrading enzymes (heparinases) assist in the breakdown of ECM topromote tumor growth, angiogenesis and metastasis. Other evidence suchas the recent cloning of tumor heparinase genes has led to the paradigmthat, the expression of HLGAG degrading enzymes represents a ‘switch’from a primary tumor to a metastatic disease state.

In surprising contrast to the findings of the prior art, it has now beendiscovered according to the invention that not only is the prior artincorrect in stating that HLGAG degrading enzymes may contribute totumor growth and metastasis, but in fact that certain HLGAG degradingenzymes and HLGAG fragments (including LMWH compositions generated byheparinase III), actually, are very effective in inhibiting cancer cellgrowth and metastasis. In particular, it has been discovered thatheparinases having similar functional activity to native heparinase IIIprevent in vivo tumor growth and metastasis. It has also been discoveredthat the enzymatic products of heparinase III (HLGAG fragments and LMWH)are useful for preventing tumor growth and metastasis.

The Examples section provides in vitro and in vivo data demonstratingthe effectiveness of the heparinases in preventing tumor growth andmetastasis. Using two different animal models of cancer, B16BL6 and LLC,strikingly similar data was obtained, indicating an important role forHLGAGs in tumor growth and metastasis. The data also demonstrated thedifferential effects of heparinases I and III, and the HLGAG fragmentsgenerated by these heparinases on physiological processes. Heparinase Iwas unable to prevent cancer cell proliferation or metastasis,indicating that the effects are specific to heparinase III andfunctional variants thereof. These results are consistent with theunique specificities of heparinases, and hence the distinctoligosaccharide products they generate. Additionally, the datademonstrated that HLGAG fragments for one cell type were able toinfluence effects on another cell type, strongly indicating theinvolvement of specific sequences of HLGAG in modulating effects ontumor growth and metastasis.

Thus, the invention includes methods for treating or preventing tumorformation and/or metastasis by administering to a subject a heparinaseIII molecule (native or modified) and/or therapeutic HLGAG fragments(including LMWH).

The heparinases useful in this aspect of the invention include nativeheparinase III, modified heparinase III and modified heparinases havingthe functional activity of heparinase III. “Native heparinase III” asused herein refers to the naturally occurring heparinase III molecule inan isolated form. The sequence of the naturally occurring molecule fromF. heparinum is provided as SEQ ID NO.: 1 (nucleic acid sequence) and 2(amino acid sequence), and has been extensively described in artincluding in issued patents. An isolated molecule is a molecule that issubstantially pure and is free of other substances with which it isordinarily found in nature or in vivo systems to an extent practical andappropriate for its intended use. In particular, the molecular speciesare sufficiently pure and are sufficiently free from other biologicalconstituents of host cells so as to be useful in, for example, producingpharmaceutical preparations or sequencing if the molecular species is anucleic acid, peptide, or polysaccharide. Because an isolated molecularspecies of the invention may be admixed with apharmaceutically-acceptable carrier in a pharmaceutical preparation, themolecular species may comprise only a small percentage by weight of thepreparation. The molecular species is nonetheless substantially pure inthat it has been substantially separated from the substances with whichit may be associated in living systems.

A “modified heparinase III” as used herein is any heparinase IIImolecule which has at least one mutation, deletion or substitution,compared to native heparinase III but which retains the ability toenzymatically cleave heparan sulfate. These include the particularmodified heparinases described herein as well as any other modifiedheparinase having the appropriate function. These can be identified bythose of ordinary skill in the art using the methods described above orin the examples section. For instance, the modified heparinase III mayhave a simple conservative substitution within a region of the moleculewhich is not critical for enzymatic activity or folding and thus whichhas no effect on the ability of the heparinase to cleave the substrate.Additionally, substitutions such as the histidine substitutionsdescribed herein which influence the enzymatic activity or productprofile of the heparinase but which still retain some enzymatic activityare also useful for this aspect of the invention because they are stillable to cleave heparan sulfate. The two histidine mutations (His 295 andHis 510) which lost all activity, however, are not useful in this aspectof the invention. (These two mutants have other utilities, such ascompetitive inhibitors.)

The term “modified heparinases having functional activity of heparinaseIII” as used herein refers to heparinases other than heparinase IIIwhich have been modified such that they are enzymatically active towardsheparan sulfate but only have minimal or no activity towards heparin.For instance, mutation of Cys³⁴⁸ of heparinase II, a residue which isinvolved in heparin binding, causes the heparinase II to have a reducedenzymatic activity with respect to heparin. This modification produces amodified heparinase II which becomes exclusively a heparan sulfatedegrading enzyme. Additionally, when histidine 440 is mutated inheparinase III, a modified heparinase III is produced which has reducedenzymatic activity with respect to heparin but which displays nearly thesame enzymatic activity as native heparinase III when heparan sulfate isused as the substrate. Mutation of histidines 451, 238, and 579 ofheparinase II produces modified heparinase II molecules having reducedenzymatic activity with respect to heparan sulfate. Thus modifiedheparinase II molecules in which the Cys³⁴⁸ or His⁴⁴⁰ is mutated are“modified heparinases having functional activity of heparinase III”according to the invention, whereas heparinases in which histidines 451,238, or 579 have been mutated are not within this class of molecules.

The invention also contemplates the use of therapeutic HLGAGs for thetreatment and prevention of tumor cell proliferation and metastasis. Atherapeutic HLGAG fragment as used herein refers to a molecule ormolecules which are pieces or fragments of an HLGAG that have beenidentified through the use of the native heparinase III, modifiedheparinase III and modified heparinases having the functional activityof heparinase III described above. HLGAG fragments also include lowmolecular weight heparins (LMWHs). The compositional analysis of sometherapeutic HLGAGs is described below in the Examples section.

The invention also encompasses screening assays for identifyingtherapeutic HLGAG fragments for the treatment of a tumor and forpreventing metastasis. The assays are accomplished by treating a tumoror isolated tumor cells with heparinase III, native or modified andisolating the resultant HLGAG fragments. Surprisingly, these HLGAGfragments have therapeutic activity in the prevention of tumor cellproliferation and metastasis. As described in more detail in theExamples section, these HLGAG fragments are useful as therapeutic agentsfor the treatment of the tumor cells from which they were generated aswell as other tumors. Thus the invention encompasses individualizedtherapies, in which a tumor or portion of a tumor is isolated from asubject and used to prepare the therapeutic HLGAG fragments. Thesetherapeutic fragments can be re-administered to the subject to protectthe subject from further tumor cell proliferation or metastasis or fromthe initiation of metastasis if the tumor is not yet metastatic.Alternatively the fragments can be used in a different subject havingthe same type or tumor or a different type of tumor.

The term “therapeutic HLGAG fragment” as used herein refers to an HLGAGwhich has therapeutic activity in that it prevents the proliferationand/or metastasis of a tumor cell. Such compounds can be generated usingheparinase III to produce therapeutic fragments or they can besynthesized de novo. Putative HLGAG fragments can be tested fortherapeutic activity using any of the assays described herein or knownin the art. Thus the therapeutic HLGAG fragment may be a synthetic HLGAGfragment generated based on the sequence of the HLGAG fragmentidentified when the tumor is contacted with heparinase III, or havingminor variations which do not interfere with the activity of thecompound. Alternatively the therapeutic HLGAG fragment may be anisolated HLGAG fragment produced when the tumor is contacted withheparinase III.

The invention is useful for treating and/or preventing tumor cellproliferation or metastasis in a subject. The terms “prevent” and“preventing” as used herein refer to inhibiting completely or partiallythe proliferation or metastasis of a cancer or tumor cell, as well asinhibiting any increase in the proliferation or metastasis of a canceror tumor cell.

A “subject having a cancer” is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A “subject at risk of having a cancer” as used herein is a subject whohas a high probability of developing cancer. These subjects include, forinstance, subjects having a genetic abnormality, the presence of whichhas been demonstrated to have a correlative relation to a higherlikelihood of developing a cancer and subjects exposed to cancer causingagents such as tobacco, asbestos, or other chemical toxins, or a subjectwho has previously been treated for cancer and is in apparent remission.When a subject at risk of developing a cancer is treated with aheparinase III the subject may be able to kill the cancer cells as theydevelop.

Effective amounts of the native heparinase III, modified heparinases, ortherapeutic HLGAGs of the invention are administered to subjects in needof such treatment. Effective amounts are those amounts which will resultin a desired reduction in cellular proliferation or metastasis withoutcausing other medically unacceptable side effects. Such amounts can bedetermined with no more than routine experimentation. It is believedthat doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram,depending upon the mode of administration, will be effective. Theabsolute amount will depend upon a variety of factors (including whetherthe administration is in conjunction with other methods of treatment,the number of doses and individual patient parameters including age,physical condition, size and weight) and can be determined with routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment. Themode of administration may be any medically acceptable mode includingoral, subcutaneous, intravenous, etc.

In some aspects of the invention the effective amount of heparinase IIIis that amount effective to prevent invasion of a tumor cell across abarrier. The invasion and metastasis of cancer is a complex processwhich involves changes in cell adhesion properties which allow atransformed cell to invade and migrate through the extracellular matrix(ECM) and acquire anchorage-independent growth properties. Liotta, L.A., et al., Cell 64:327-336 (1991). Some of these changes occur at focaladhesions, which are cell/ECM contact points containingmembrane-associated, cytoskeletal, and intracellular signalingmolecules. Metastatic disease occurs when the disseminated foci of tumorcells seed a tissue which supports their growth and propagation, andthis secondary spread of tumor cells is responsible for the morbidityand mortality associated with the majority of cancers. Thus the term“metastasis” as used herein refers to the invasion and migration oftumor cells away from the primary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro ora natural barrier in vivo. In vitro barriers include e but are notlimited to extracellular matrix coated membranes, such as Matrigel. Thusthe heparinase compositions can be tested for their ability to inhibittumor cell invasion in a Matrigel invasion assay system as described indetail by Parish, C. R., et al., “A Basement-Membrane Permeability Assaywhich Correlates with the Metastatic Potential of Tumour Cells,” Int. J.Cancer (1992) 52:378-383. Matrigel is a reconstituted basement membranecontaining type IV collagen, laminin, heparan sulfate proteoglycans suchas perlecan, which bind to and localize bFGF, vitronectin as well astransforming growth factor-β (TGF-β), urokinase-type plasminogenactivator (uPA), tissue plasminogen activator (tPA), and the serpinknown as plasminogen activator inhibitor type 1 (PAI-1). Other in vitroand in vivo assays for metastasis have been described in the prior art,see, e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which isincorporated by reference. An in vivo barrier refers to a cellularbarrier present in the body of a subject.

In general, when administered for therapeutic purposes, the formulationsof the invention are applied in pharmaceutically acceptable solutions.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medicaluse, which comprise native heparinase III, modified heparinases of theinvention, or therapeutic HLGAG fragments together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. The term “pharmaceutically-acceptable carrier” as usedherein, and described more fully below, means one or more compatiblesolid or liquid filler, dilutants or encapsulating substances which aresuitable for administration to a human or other animal. In the presentinvention, the term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The components of thepharmaceutical compositions also are capable of being commingled withthe modified heparinases of the present invention, and with each other,in a manner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficiency.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular modified heparinaseselected, the particular condition being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofan immune response without causing clinically unacceptable adverseeffects. A preferred mode of administration is a parenteral route. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intraperitoneal, intra sternal injection or infusiontechniques. Other modes of administration include oral, mucosal, rectal,vaginal, sublingual, intranasal, intratracheal, inhalation, ocular,transdermal, etc.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active modified heparinaseinto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the polymer into association with a liquid carrier,a finely divided solid carrier, or both, and then, if necessary, shapingthe product. The polymer may be stored lyophilized.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the heparinases of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. Specific examplesinclude, but are not limited to: (a) erosional systems in which thepolysaccharide is contained in a form within a matrix, found in U.S.Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); andU.S. Pat. Nos. 4,748,034 and 5,239,660 (Leonard) and (b) diffusionalsystems in which an active component permeates at a controlled ratethrough a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) andU.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardwaredelivery system can be used, some of which are adapted for implantation.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse,cow, pig.

When administered to a patient undergoing cancer treatment, theheparinase III compounds may be administered in cocktails containingother anti-cancer agents. The compounds may also be administered incocktails containing agents that treat the side-effects of radiationtherapy, such as anti-emetics, radiation protectants, etc.

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta- I a; Interferon Gamma- I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

The heparinase III compounds may also be linked to a targeting molecule.A targeting molecule is any molecule or compound which is specific for aparticular cell or tissue and which can be used to direct the heparinaseIII to the cell or tissue. Preferably the targeting molecule is amolecule which specifically interacts with a cancer cell or a tumor. Forinstance, the targeting molecule may be a protein or other type ofmolecule that recognizes and specifically interacts with a tumorantigen.

Tumor-antigens include Melan-A/M A RT -1, Dipeptidyl peptidase IV(DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b,Colorectal associated antigen (CRC)-C017-1A/GA733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1,Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1,PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100^(Pmel117), PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1,SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatouspolyposis coli protein (APC), fodrin, P1A, Connexin 37, Ig-idiotype,p15, gp75, GM2 and GD2 gangliosides, viral products such as humanpapilloma virus proteins, Smad family of tumor antigens, lmp-1,EBV-encoded nuclear antigen (EBNA)-1, and c-erbB-2.

Examples of tumor antigens which bind to either or both MHC class I andMHC class II molecules, see the following references: Coulie, Stem Cells13:393-403, 1995; Traversari et al., J. Exp. Med. 176:1453-1457, 1992;Chaux et al., J. Immunol. 163:2928-2936, 1999; Fujie et al., Int. J.Cancer 80:169-172, 1999; Tanzarella et al., Cancer Res. 59:2668-2674,1999; van der Bruggen et al., Eur. J. Immunol. 24:2134-2140, 1994; Chauxet al., J. Exp. Med. 189:767-778, 1999; Kawashima et al, Hum. Immunol.59:1-14, 1998; Tahara et al., Clin. Cancer Res. 5:2236-2241, 1999;Gaugler et al., J. Exp. Med. 179:921-930, 1994; van der Bruggen et al.,Eur. J. Immunol. 24:3038-3043, 1994; Tanaka et al., Cancer Res.57:4465-4468, 1997; Oiso et al., Int. J. Cancer 81:387-394, 1999; Hermanet al., Immunogenetics 43:377-383, 1996; Manici et al., J. Exp. Med.189:871-876, 1999; Duffour et al., Eur. J. Immunol. 29:3329-3337, 1999;Zom et al., Eur. J. Immunol. 29:602-607, 1999; Huang et al., J. Immunol.162:6849-6854, 1999; Boël et al., Immunity 2:167-175, 1995; Van denEynde et al., J. Exp. Med. 182:689-698, 1995; De Backer et al., CancerRes. 59:3157-3165, 1999; Jäger et al., J. Exp. Med. 187:265-270, 1998;Wang et al., J. Immunol. 161:3596-3606, 1998; Aamoudse et al., Int. J.Cancer 82:442-448, 1999; Guilloux et al., J. Exp. Med. 183:1173-1183,1996; Lupetti et al., J. Exp. Med. 188:1005-1016, 1998; Wölfel et al.,Eur. J. Immunol. 24:759-764, 1994; Skipper et al., J. Exp. Med.183:527-534, 1996; Kang et al., J. Immunol. 155:1343-1348, 1995; Morelet al., Int. J. Cancer 83:755-759, 1999; Brichard et al., Eur. J.Immunol. 26:224-230, 1996; Kittlesen et al., J. Immunol. 160:2099-2106,1998; Kawakami et al., J. Immunol. 161:6985-6992, 1998; Topalian et al.,J. Exp. Med. 183:1965-1971, 1996; Kobayashi et al., Cancer Research58:296-301, 1998; Kawakami et al., J. Immunol. 154:3961-3968, 1995; Tsaiet al., J. Immunol. 158:1796-1802, 1997; Cox et al., Science264:716-719, 1994; Kawakami et al., Proc. Natl. Acad Sci. USA91:6458-6462, 1994; Skipper et al., J. Immunol. 157:5027-5033, 1996;Robbins et al., J. Immunol. 159:303-308, 1997; Castelli et al, J.Immunol. 162:1739-1748, 1999; Kawakami et al., J. Exp. Med. 180:347-352,1994; Castelli et al., J. Exp. Med. 181:363-368, 1995; Schneider et al.,Int. J. Cancer 75:451-458, 1998; Wang et al., J. Exp. Med.183:1131-1140, 1996; Wang et al., J. Exp. Med. 184:2207-2216, 1996;Parkhurst et al., Cancer Research 58:4895-4901, 1998; Tsang et al., J.Natl Cancer Inst 87:982-990, 1995; Correale et al., J Natl Cancer Inst89:293-300, 1997; Coulie et al., Proc. Natl. Acad. Sci. USA92:7976-7980, 1995; Wölfel et al., Science 269:1281-1284, 1995; Robbinset al., J. Exp. Med. 183:1185-1192, 1996; Brändle et al., J. Exp. Med.183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996;Mandruzzato et al., J. Exp. Med. 186:785-793, 1997; Guéguen et al., J.Immunol. 160:6188-6194, 1998; Gjertsen et al., Int. J. Cancer72:784-790, 1997; Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiariet al., Cancer Res. 59:5785-5792, 1999; Hogan et al., Cancer Res.58:5144-5150, 1998; Pieper et al., J. Exp. Med. 189:757-765, 1999; Wanget al., Science 284:1351-1354, 1999; Fisk et al., J. Exp. Med.181:2109-2117, 1995; Brossart et al., Cancer Res. 58:732-736, 1998;Röpke et al., Proc. Natl. Acad. Sci. USA 93:14704-14707, 1996; Ikeda etal., Immunity 6:199-208, 1997; Ronsin et al., J. Immunol. 163:483-490,1999; Vonderheide et al., Immunity 10:673-679,1999. These antigens aswell as others are disclosed in PCT Application PC/US98/18601.

One of ordinary skill in the art, in light of the present disclosure, isenabled to produce substantially pure preparations of any of the nativeor modified heparinases by standard technology, including recombinanttechnology, direct synthesis, mutagenesis, etc. For instance, usingrecombinant technology one may substitute appropriate codons in SEQ IDNO: 1 to produce the desired amino acid substitutions by standardsite-directed mutagenesis techniques. Obviously, one may also use anysequence which differs from SEQ ID NO: 1 only due to the degeneracy ofthe genetic code as the starting point for site directed mutagenesis.The mutated nucleic acid sequence may then be ligated into anappropriate expression vector and expressed in a host such as F.heparinum or E. coli. The resultant modified heparinase may then bepurified by techniques well known in the art, including those disclosedbelow and in Sasisekharan, et al. (1993). As used herein, the term“substantially pure” means that the proteins are essentially free ofother substances to an extent practical and appropriate for theirintended use. In particular, the proteins are sufficiently pure and aresufficiently free from other biological constituents of their hostscells so as to be useful in, for example, protein sequencing, orproducing pharmaceutical preparations.

In another set of embodiments an isolated nucleic acid encoding thesubstantially pure modified heparinase of the invention is provided. Asused herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein the codingsequences are operably joined to regulatory sequences. Two DNA sequencesare said to be operably joined if induction of a promoter in the 5′regulatory sequences results in the transcription of the coding sequenceand if the nature of the linkage between the two DNA sequences does not(1) result in the introduction of a frame-shift mutation, (2) interferewith the ability of the promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability of thecorresponding RNA transcript to be translated into a protein. Thus, apromoter region would be operably joined to a coding sequence if thepromoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Promoters may beconstitutive or inducible. Regulatory sequences may also includeenhancer sequences or upstream activator sequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium, or just a single time per host as thehost reproduces by mitosis. In the case of phage, replication may occuractively during a lytic phase or passively during a lysogenic phase. Anexpression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., β-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells, hosts,colonies or plaques. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

As used herein, the term “stringent conditions” refers to parametersknown to those skilled in the art. One example of stringent conditionsis hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25mM NaH₂PO₄ (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodiumchloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; andEDTA is ethylene diamine tetra acetic acid. There are other conditions,reagents, and so forth which can be used, which result in the samedegree of stringency. A skilled artisan will be familiar with suchconditions, and thus they are not given here. The skilled artisan alsois familiar with the methodology for screening cells for expression ofsuch molecules, which then are routinely isolated, followed by isolationof the pertinent nucleic acid. Thus, homologs and alleles of thesubstantially pure modified heparinases of the invention, as well asnucleic acids encoding the same, may be obtained routinely, and theinvention is not intended to be limited to the specific sequencesdisclosed.

For prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors include pBR322, pUC18,pUC19 and the like; suitable phage or bacteriophage vectors includeλgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo,pKRC and the like. Preferably, the selected vector of the presentinvention has the capacity to autonomously replicate in the selectedhost cell. Useful prokaryotic hosts include bacteria such as E. coli,Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and the like.

To express the substantially pure modified heparinases of the inventionin a prokaryotic cell, it is necessary to operably join the nucleic acidsequence of a substantially pure modified heparinase of the invention toa functional prokaryotic promoter. Such promoter may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ, lacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)) and the ζ-28-specific promoters of B subtilis(Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).

Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); andGottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the encoding sequence. Such ribosomebinding sites are disclosed, for example, by Gold et al. (Ann. Rev.Microbiol. 35:365-404 (1981)).

Because prokaryotic cells will not produce the modified heparinases ofthe invention with normal eukaryotic glycosylation, expression of themodified heparinases of the invention of the invention by eukaryotichosts is possible when glycosylation is desired. Preferred eukaryotichosts include, for example, yeast, fungi, insect cells, and mammaliancells, either in vivo or in tissue culture. Mammalian cells which may beuseful as hosts include HeLa cells, cells of fibroblast origin such asVERO or CHO-K1, or cells of lymphoid origin, such as the hybridomaSP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferredmammalian host cells include SP2/0 and J558L, as well as neuroblastomacell lines such as IMR 332 that may provide better capacities forcorrect post-translational processing. Embryonic cells and mature cellsof a transplantable organ also are useful according to some aspects ofthe invention.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.

Another preferred host is an insect cell, for example in Drosophilalarvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, Science 240:1453-1459(1988)). Alternatively, baculovirus vectors can be engineered to expresslarge amounts of the modified heparinases of the invention in insectscells (Jasny, Science 238:1653 (1987); Miller et al., In: GeneticEngineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp.277-297).

Any of a series of yeast gene sequence expression systems whichincorporate promoter and termination elements from the genes coding forglycolytic enzymes and which are produced in large quantities when theyeast are grown in media rich in glucose may also be utilized. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provide substantial advantages inthat they can also carry out post-translational peptide modifications. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognize leadersequences on cloned mammalian gene sequence products and secretepeptides bearing leader sequences (i.e., pre-peptides).

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or which are subject to chemical (such asmetabolite) regulation.

As discussed above, expression of the modified heparinases of theinvention in eukaryotic hosts requires the use of eukaryotic regulatoryregions. Such regions will, in general, include a promoter regionsufficient to direct the initiation of RNA synthesis. Preferredeukaryotic promoters include, for example, the promoter of the mousemetallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982);Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the modified heparinases of the inventiondoes not contain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the modified heparinases of the invention codingsequence) or a frame-shift mutation (if the AUG codon is not in the samereading frame as the modified heparinases of the invention codingsequence).

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay, for example, provide for prototrophy to an auxotrophic host or mayconfer biocide resistance to, e.g., antibiotics, heavy metals, or thelike. The selectable marker gene sequence can either be directly linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection. Additional elements may also be needed foroptimal synthesis of the modified heparinases of the invention mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, Molec.Cell. Biol. 3:280 (1983).

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184, and πVX. Such plasmids are, for example, disclosed bySambrook, et al. (Molecular Cloning: A Laboratory Manual, secondedition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring HarborLaboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127, andthe like. Such plasmids are disclosed by Gryczan (In: The MolecularBiology of the Bacilli, Academic Press, NY (1982), pp. 307-329).Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J.Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages suchas φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect.Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274(1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollonet al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608 (1980)). Other preferred eukaryoticvectors are viral vectors. For example, and not by way of limitation,the pox virus, herpes virus, adenovirus and various retroviruses may beemployed. The viral vectors may include either DNA or RNA viruses tocause expression of the insert DNA or insert RNA. Additionally, DNA orRNA encoding the modified heparinases of the invention polypeptides maybe directly injected into cells or may be impelled through cellmembranes after being adhered to microparticles.

Once the vector or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means, i.e.,transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,and the like. After the introduction of the vector, recipient cells aregrown in a selective medium, which selects for the growth ofvector-containing cells. Expression of the cloned gene sequence(s)results in the production of the modified heparinases of the invention.This can take place in the transformed cells as such, or following theinduction of these cells to differentiate (for example, byadministration of bromodeoxyuracil to neuroblastoma cells or the like).

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Materials and Methods

Chemicals and Materials. Hydroxylamine hydrochloride and urea were fromEM Science (Gibbstown, N.J.). The chemical modification reagentdiethylpyrocarbonate (DEPC) was purchased from Sigma and used asreceived (Milwaukee, Wis.). Heparan sulfate was purchased from CelsusLaboratories (Cincinnati, Ohio). Lys-C from Lysobacter enzymogenes (EC3.4.21.50) was from Roche Molecular Biochemicals (Indianapolis, Ind.).Heparinase III from Flavobacterium heparinum (EC 4.2.2.8) was purifiedas described previously (Godavarti, R., Cooney, C. L., Langer, R., andSasisekharan, R. (1996) Biochemistry 35, 6846-52 and Lohse, D. andLinhardt, R. J. (1992) J. Biol. Chem. 267, 781-87) and was a gift fromIBEX Technologies (Montreal, Canada).

Heparinase III Activity Assay

The activity of heparinase III was measured using a UV 232 nm assaysimilar to those reported for heparinase I and heparinase II (Godavarti,R., Cooney, C. L., Langer, R., and Sasisekharan, R. (1996) Biochemistry35, 6846-52; Shriver, Z., Hu, Y., and Sasisekharan, R. (1998) J. Biol.Chem. 273, 10160-67; and Lohse, D. and Linhardt, R. J. (1992) J. Biol.Chem. 267, 781-87). Briefly, the increase in absorbance at 232 nm as afunction of time was monitored under saturating substrate conditions.All assays were performed with heparan sulfate at a concentration of 2mg/ml in 50 mM sodium phosphate, pH 7.6. The temperature for enzymaticactivity measurements was kept constant at 35° C.

Chemical Modification of Heparinase III with DEPC

(A) DEPC Inactivation of Heparinase III. At pH values ranging from 6.0to 8.0, heparinase III (50 μg/mL) was incubated with DEPC in 50 mMsodium phosphate buffer at 25° C. The DEPC stock solution (6.9 M) wasdiluted with ethanol. Control reactions contained an equivalent amountof ethanol instead of DEPC and were found to not affect enzymaticactivity over the experimental time range. At each pH, three reactionswere run using different concentrations of DEPC, ranging from 50 μM to2.5 mM. At fixed time intervals, 25 μL aliquots were withdrawn from thereaction mixtures for the UV 232 nm activity assay.

The kinetics of DEPC inactivation of heparinase III was determined byplotting the natural log of percent activity versus an adjusted timeterm (to account for the decomposition of DEPC). Briefly, this adjustedtime term (t′) was calculated according to the following equation:$t^{\prime} = \frac{1 - e^{k^{\prime}t}}{k^{\prime}}$

In this equation, k′ is the first order rate constant for DEPChydrolysis and t is the measured time after addition of DEPC to theheparinase III solution. At each pH, the order of the reaction in DEPCwas determined by plotting the observed rate constants of inactivationat each pH vs. log [DEPC]. The slope of this graph is n, the order ofthe reaction with respect to DEPC (Lundblad, R. (1995) Techniques inProtein Modification, CRC Press, Boca Raton).

(B) Reactivation of DEPC—Modified Enzyme with Hydroxylamine. Similar towhat was completed with heparinase I and II (Godavarti, R., Cooney, C.L., Langer, R., and Sasisekharan, R. (1996) Biochemistry 35, 6846-52 andShriver, Z., Hu, Y., and Sasisekharan, R. (1998) J. Biol. Chem. 273,10160-67), heparinase III (50 μg/mL) was incubated with 0.97 mM DEPC, pH6.5 until its enzymatic activity was reduced to 50% of its initialvalue. Hydroxylamine was then immediately added to the reaction mixtureto a final concentration of 0.5 M. The reaction was incubated at roomtemperature for 6 hours. Aliquots were withdrawn every hour for theactivity assay. The control mixture contained no DEPC but the sameconcentration of hydroxylamine to account for the loss of nonspecificactivity. The ratio of the activity of the reaction mixture over theactivity of the control was calculated to determine recovery ofenzymatic activity.

(C) Substrate Protection of Heparinase III against DEPC Inactivation.Heparinase III (50 μg/mL) was pre-incubated with 2 mg/mL heparan sulfatein 50 mM sodium phosphate, pH 7.6 for 30 minutes prior to the additionof 1.5 mM DEPC. A control reaction with no prior incubation of substratewas also completed. The time course of inactivation for both wasdetermined using the heparinase III activity assay.

(D) Quantification of the Number of Histidines Modified by DEPC. Theextent of modification of an enzyme by DEPC can be determined bymonitoring the formation of the N-carbethoxylhistidyl adduct at 240 nm.At time zero, 1.5 mM DEPC was added to the cuvette containing heparinaseIII in sodium phosphate buffer, pH 7.0. The change in absorbance at 240nm was monitored every minute for 10 min. The number of modifiedresidues was determined using a molar extinction coefficient of 3,200M⁻¹cm⁻¹ (Lundblad, R. (1995) Techniques in Protein Modification, CRCPress, Boca Raton).

Peptide Mapping Studies

To determine which histidine residues were modified by DEPC, mappingstudies using the protease Lys-C were completed. Heparinase III (1nmole) was incubated with 4 mM DEPC for fifteen minutes, denatured with6.5 M urea at 55° C. and diluted with water. Subsequently, thedenatured, modified heparinase III was digested with Lys-C.

Peptides derived from heparinase III digestion were separated by reversephase high performance liquid chromatography (RPHPLC) and monitored at210, 277, and 240 nm. Peptide peaks not present in the control digestwere collected and sequenced using an Applied Biosystems Sequencer model477 with an on-line model 120 PTH amino acid analyzer (BiopolymersLaboratory, MIT).

Site-directed Mutagenesis

Each of the thirteen histidine residues of heparinase III was mutated toalanine using overlap extension PCR for 15 cycles (Higuchi, R. (1990) inPCR Protocols. A Guide to Methods and Applications (Innis, M., Gelfand,D. H., Sninsky, J. J., and White, T. J., Ed.) pp 177-83, Academic Press,San Diego). The PCR reactions were separated on a low-melt Agarose geland the band corresponding to the proper molecular weight was excised.The DNA was extracted from the gel using a Gel Purification Kit (QIAGEN,Valencia, Calif.) and the insert was subcloned into pCR 2.1 (Invitrogen,Carlsbad, Calif.). The validity of all the point mutations and theintegrity of the rest of the gene were verified by sequencing. Thethirteen mutant heparinase III sequences were prepared in pCR2. 1 usinga Miniprep kit (QIAGEN, Valencia, Calif.) and cloned using Nde I/BamH I(New England Biolabs, Beverly, Mass.) into pET-15b (Novagen, Madison,Wis.) for expression. The pET-15b plasmid contains a NH₂-terminalHis-Tag for Ni²⁺-column purification. Recombinant heparinase III wasalso expressed and compared to the native heparinase III isolateddirectly from Flavobacterium heparinum.

Expression, Isolation, and Purification of r-heparinase III and Mutantsin E. coli

Overnight cultures of Luria-Bertani (LB) broth (5 ml) containing 0.02mg/ml ampicillin (amp) were used to inoculate 500 ml LB/amp cultures atan initial OD₆₀₀ of 0.1. The cultures were induced with 1 mMisopropyl-B-D-thiogalactopyranoside (IPTG) in mid-log phase (OD₆₀₀0.7-0.9) and incubated for another hour at 37° C. To harvest the cells,the cultures were spun at 5,000 rpm and the supernatant was discarded.

The cell pellet was re-suspended in 20 mM Tris, 500 mM NaCl, 5 mMimmidazole-HCl, pH 7.9 (1/50 of the initial culture volume). There-suspended cells were placed on ice and sonicated as describedpreviously (Ernst, S., Venkataraman, G., Winkler, S., Godavarti, R.,Langer, R., Cooney, C. L., and Sasisekharan, R. (1996) Biochem. J. 315,589-97). The soluble protein of the cell lysate was isolated bycentrifugation at 12,000 rpm for 20 min. at 4° C. The supernant wasfiltered through a 0.45 μm filter and loaded onto a nickel column usinga Biocad Perfusion Chromatography system (PerSeptive Biosystems,Framingham, Mass.). The column was washed and the protein wassubsequently eluted in 20 mM Tris, 500 mM NaCl, 500 mM immidazole-HCl,pH 7.9. SDS-polyacrylamide gel electrophoresis analysis using precast12% gels, the Mini-Protean II apparatus, and the Silver Stain Pus kit(Bio-Rad, Hercules, Calif.) was performed to determine the concentrationand purity of the individual proteins.

HPLC Analysis of Saccharide Products of Heparinase III Activity

Exhaustive digests of 3 mg/ml heparan sulfate in 50 mM sodium phosphatebuffer, pH 7.6 were performed overnight at 37° C. for each of themutants (20 μg protein). The reactions were loaded onto a Spherisorb S5SAX column (Waters) and eluted using a linear gradient of 0.2-1.0 MNaCl, pH 3.5. The products were monitored at 232 nm and each of themajor peaks was collected. To determine their composition, the collectedfractions were analyzed by capillary electrophoresis and identified bycomigration with known standards.

Circular Dichroism (CD)

Recombinantly expressed heparinase III and the heparinase III mutants,H295A and H510A were concentrated and buffer-exchanged into 50 mM sodiumphosphate, pH 7.0 using a Centricon 30 Filter (Millipore, Watertown,Mass.). CD spectra were collected on an Aviv 62DS spectropolarimeterequipped with a thermostatic temperature controller and interfaced to anIBM microcomputer. Measurements were performed in a quartz cell with a 1mm path length. Spectra were recorded at 25° C., in an average of 10scans between 205 and 260 nm, with a 1.0 nm bandwidth and a scan rate of3 nm/min. CD band intensities are expressed as molar ellipticities,θ_(M), in degrees·cm²·dmol⁻¹.

Transfection of B16 cells:

B16BL6 melanoma cells were transfected with antisense 2OST in pcDNA3.1.Stable transfectant clones were selected with G418 and propagated. Thesuccess of transfection was confirmed with PCR screening of transfectedcells.

In vitro invasion assay:

10⁵ of B16BL6 and B16BL6 transfectant were loaded onto inserts coatedwith 15 ug of Matrigel. MEM-α with 40 ng/ml of bFGF was used aschemoattractant. After 20 hours incubation at 37° C., inserts were fixedand stained. Unmigrated cells were removed and migrated cells werecounted under light microscope.

In vivo primary tumor growth:

4×10⁵ B16BL6, transfected and untransfected respectively, wereinoculated subcutaneously to the flank of nude mice. Measurement oftumor size started on day 10 after tumor cell injection. and mice wereeuthanized on day 16 after the injection.

In vivo lung metastasis:

2×10⁵ B16BL6 in 0.2 ml PBS, transfected and untransfected respectively,were injected via tail vein of C57BL6 mice. 13 days later, the mice wereeuthanized, lungs harvested and analyzed.

Compositional Analysis of HLGAGs resulting from heparinase treatment ofB16 cells:

B16 cells were treated with either heparinase I, III or PBS. Thesupernatant was collected, boiled, and filtered through a 0.45 μmfilter. This sample was then subjected to fractionation using acentricon spin column with a nominal molecular weight cutoff of 5 kDa.The retentate was exchanged into water and concentrated 50-fold bylyophilization.

Compositional analysis of oligosaccharides was completed by exhaustivedigest of the high molecular weight fraction with heparinases I-III. To9 μL of aqueous oligosaccharide was added 1 mU of heparinases I-III in25 mM sodium acetate, 2 mM calcium chloride buffer at pH 7.0. Thereaction was allowed to proceed at 37° C. overnight after which CEanalysis was completed.

Compositional analysis was completed on a Hewlett Packard 3D CE unit byusing uncoated fused silica capillaries (i.d. 75 μM). Analytes weremeasured using an extended path length capillary. The electrolyte was 50mM tris/phosphate pH 2.5. Separations were carried out at 30 kV withreverse polarity. Assignments and quantification of disaccharides weremade by comparison with known standards.

Results

Example 1

DEPC Inactivates Heparinase III

As a first step towards identifying histidines that are critical for theenzymatic activity of heparinase III, the effect of the modificationreagent DEPC on the enzymatic activity of heparinase III was determined.DEPC is a common reagent used for the determination of catalyticallycritical histidines in enzymes. As stated in early publications(Godavarti, R., Cooney, C. L., Langer, R., and Sasisekharan, R. (1996)Biochemistry 35, 6846-52 and Shriver, Z., Hu, Y., and Sasisekharan, R.(1998) J. Biol. Chem. 273, 10160-67), DEPC is useful for thedetermination of catalytically critical histidines, however care needsto be taken to ensure that other nucleophilic residues, namelytyrosines, lysines, and cysteines are not modified.

Heparinase III was incubated with 0.31 (□), 0.54 (●), 0.97 (∘), 1.5 (▴),1.9 (Δ) mM DEPC at pH 6.5 and at 25° C. (shown in inset of FIG. 1). Thenatural log of the percent activity remaining was plotted versus anadjusted time term (t′) to account for the decomposition of DEPC. Theslope of each of the lines at the various DEPC concentrations representsthe pseudo-first order rate constants of inactivation. Plotting thesepseudo-first order rate constants versus the respective DEPCconcentrations yields a second-order rate constant of inactivation of0.20±0.04 mM⁻¹min⁻¹.

For heparinase III, similar to heparinase I and II, DEPC was found toinhibit in a dose-dependent fashion. A measured second order rateconstant of 0.20±0.04 min⁻¹mM⁻¹ is obtained by varying the concentrationof the inhibitor. Consistent with this reaction being first order inboth heparinase III and DEPC, a plot of k_(inact) vs. log [DEPC] yieldeda line with a slope of 1 (FIG. 1).

The fact that DEPC inactivates heparinase III in a pseudo-first order,dose-dependent manner suggests that DEPC is directly modifying a residueinvolved in the catalytic degradation of heparan sulfate. Thesecond-order rate constant of inactivation (0.20±0.04 min⁻¹mM⁻¹) alsosuggests that DEPC is a potent inhibitor of heparinase III function.

To ensure that the interaction of DEPC with heparinase III is throughhistidine modification, we investigated whether other nucleophilic aminoacids of heparinase III interact with DEPC. First, unlike withheparinase I or II, there is no possibility for cysteine modificationsince heparinase III contains no cysteines in its primary amino acidsequence. Furthermore, there was no loss of absorbance at 278 nm uponincubation of DEPC with heparinase III as would be expected if tyrosineswere modified. Finally, addition of hydroxylamine to DEPC-modifiedheparinase III reversed most of the inactivation indicating thatstrongly nucleophilic residues, such as lysine, were not modified byDEPC (Table 1).

TABLE 1 Hydroxylamine Reversibility of DEPC Inactivation. Time (min)Activity (%) 0 51 30 60 60 66 90 72 180 76 240 78 360 80

In an attempt to further define the interaction of DEPC with thehistidines of heparinase III, the effect of the pH on the inactivationkinetics was examined. Examination of the rate of inactivation as afunction of pH has been used to derive a pKa for a modified residue,since, in the case of histidine, the unprotonated form is much morereadily modified than is the protonated form. The pH dependence of thesecond-order rate constant of inactivation is shown in FIG. 2.(Heparinase III was incubated with 50 μM to 2.5 mM DEPC at pH's 6.0-8.0at 25° C. and the second-order rate constant of inactivation wascalculated for each pH). With heparinase III, increasing the pH of thereaction from 6.0-7.5 results in an increase in the inactivationkinetics without changing the order of the reaction (FIG. 2). However,at pH 8.0 and higher, the reaction is no longer first order in DEPC,indicating other residues (possibly lysines) are interacting with DEPCat this pH. Consistent with this interpretation, hydroxylamine is nolonger able to reverse inaction at pH 8.0. Therefore, the mappingstudies and substrate protection experiments discussed below wereconducted at pH 7.0 which maximized the reactivity while ensuring thatonly histidines were the target of DEPC modification.

DEPC-modified histidine residues in heparinase III were quantified(shown in FIG. 3). At time zero, 1.5 mM DEPC was added to a cuvettecontaining heparinase III (540 μg/mL) in sodium phosphate buffer, pH7.0. The change in absorbance at 240 nm was monitored at time intervalsfor 10 min. The number of modified histidines was calculated using aε=3200 M⁻¹cm⁻¹. At the beginning and end of the experiment, aliquots ofheparinase III were withdrawn and tested for activity. Less than 5% ofinitial activity remained after 10 minutes incubation with DEPC.

Consistent with the idea that DEPC is interacting with a histidineresidue in heparinase III, there is an increase in absorbance at 240 nmas a function of time, resulting from N-carbethoxyhistdyl derivatives.FIG. 3 shows the quantitation of the number of modified histidines. Overthe course of 10 minutes, 1.8 histidines were modified per enzymemolecule resulting in a loss of greater than 90% activity. Thus, itappears that one or possibly two histidines, modified by DEPC, result inloss of enzymatic activity for heparinase III.

Substrate protection of heparinase III inactivation by DEPC was alsoassessed (FIG. 4). Heparinase III (50 μg/mL) was incubated with 2 mg/mLheparan sulfate for 30 min. 1.5 mM DEPC was added to the reaction andtime course of inactivation was completed using the heparinase IIIactivity assay (∘). A control reaction without pre-incubation withheparan sulfate was also done (□).

Preincubation of heparinase III with heparan sulfate substrate beforeaddition of DEPC resulted in lower inactivation kinetics (FIG. 4)suggesting that the histidine(s) modified by DEPC are proximate to thesubstrate binding and/or active site of heparinase III, similar to whatwas observed for heparinase I and II (Godavarti, R., Cooney, C. L.,Langer, R., and Sasisekharan, R. (1996) Biochemistry 35, 6846-52 ANDShriver, Z., Hu, Y., and Sasisekharan, R. (1998) J. Biol. Chem. 273,10160-67).

Example 2

Peptide Mapping of the Histidine Modified by DEPC

To identify the histidine(s) modified by DEPC that resulted in the lossof enzymatic activity, DEPC-modified heparinase III was digested withLys-C. Peptides that had altered retention times and an increased inabsorbance at 240 nm as compared to a control digest were collected andsequenced (FIG. 5). Three peptides had altered retention times andincreased absorbance at 240 nm were isolated and sequenced. Two of thepeptides contained histidine 295 and one contained no modified histidineresidues.

Labeling of the DEPC-reactive histidines was completed by first reactingheparinase III with DEPC, then denaturing the protein in urea. Followingan overnight digest with Lys-C, the resultant peptides were separated byusing a 1.6%-78.4% acetonitrile gradient over 120 minutes, whichincluded a 5 min isocratic phase (1.6% acetonitrile, 0.1%trifluoroacetic acid) at the beginning of the run. Lys-C peptides weremonitored at 210, 240, and 277 nm. New peptide peaks, not present in thecontrol digest and with a marked absorbance at 277 nm were collected andsequenced. These peptides are marked with an asterisk in thechromatogram. The peptides migrating at 62 and 71 min. contained thesequence QVYADGMQFELSPIYHVAAIDIFLK (SEQ ID NO.:3) including histidine295. The other consistently labeled peptide did not contain a histidine.FIG. 5A shows C4 RPHLPLC profile of the Lys-C digest of heparinase IIIwhich was not exposed to DEPC and FIG. 5B shows the peptide profile ofheparinase III labeled with DEPC.

Example 3

Site-Directed Mutagenesis of Heparinase III

In parallel to the mapping studies and to confirm the results of thechemical modification experiments, each of the thirteen histidineresidues present in heparinase III was mutated to alanine. Therecombinant heparinase III mutant proteins were expressed, purified, andassessed for enzymatic activity towards heparan sulfate (Table 2).

Enzyme K_(M) (uM)^(a) k_(cat) (s⁻¹) wild-type 80 78 r-heparinase IIIH36A 98 86 H105A ND^(b) ND^(b) H110A 9 37 H139A 191 68 H152A 58 83 H225A80 22 H234A 75 23 H241A 16 5 H295A ND ND H424A 59 24 H469A 71 100 H510AND ND H539A 92 132 ^(a)Calculated assuming a molecular weight forheparan sulfate of 15 kDa. ^(b)Protein expression levels were too lowfor heparinase III kinetic assay.

As a control, the r-heparinase III construct without its putative signalsequence was expressed. The concentration and purity of all recombinantenzyme preparations were determined using SDS-PAGE. The recombinantlyexpressed heparinase III was also compared to the heparinase IIIisolated from F. heparinum to ensure that they were the same molecularweight. SAX analysis of exhaustive heparinase III digests of heparansulfate is shown in FIG. 6. Heparinase III (20 μg/mL) was incubated witha 4 mg/mL of heparan sulfate overnight at 37° C. The reaction was loadedonto a SAX column and the saccharide products were eluted using agradient of 0.2-1.0 M NaCl, pH 3.5 over 30 min. and monitored at 232 nm.(A) Heparan sulfate digested with heparinase III from F. heparinum. (B)Heparan sulfate digested with recombinant heparinase III. (C) Heparansulfate digested with the H295A mutant enzyme. (D) Heparan sulfatedigested with the H510A mutant enzyme. (E) Heparan sulfate digested withthe H105A mutant enzyme.

Both enzymes displayed similar kinetic activity towards heparan sulfateand yielded the same degradation profiles as determined by SAX-HPLC(FIG. 6). The products of the exhaustive digests were then analyzedusing capillary electrophoresis. The first major peak (5 min) observedin the SAX-HPLC chromatograms has a migration time that is identical toΔU-H_(NAc). The second peak (7.5 min) has a migration time that isidentical to ΔU-H_(NS) (data not shown). Thus, the heparan sulfatedegradation by recombinant heparinase III produces an identical productprofile to that of wild type heparinase III indicating that, at leastfunctionally, these enzymes are the same.

The replacement of histidine 295 and histidine 510 with alanine residuescompletely eliminated the activity of heparinase III towards heparansulfate (Table 2). The H295A and H510A mutant enzyme showed nodifferences in terms of expression level or molecular weight. However,both the kinetic data and the exhaustive digest profile for H295A andH510A suggest that the enzymes are completely inactive (FIG. 6). Nine ofthe histidine mutants (H36A, H152A, H225A, H234A, H241A, H469A, H424A,H510A and H539A) showed no significant changes in recombinant proteinyield, enzyme activity, or kinetic parameters when compared withr-heparinase III. Interestingly enough, three (H105A, H110A, and H139A)of the thirteen histidine mutants yielded much less recombinant proteinthan either recombinant heparinase III or the other mutants. Despitelower protein levels, the H110A and H139A mutant proteins were amenableto kinetic analysis whereas the H105A mutant protein was not. However,SAX-HPLC analysis of overnight heparan sulfate digests confirmed thatdespite lower levels of recombinant expression, all three of theseunder-expressed enzymes retain their catalytic activity (FIG. 6).

The recombinantly expressed heparinase III, the H295A mutant, and theH510A mutant were compared using circular dichroism (CD). Circulardichroism analysis of recombinant heparinase III and the H295A mutantenzyme is shown in FIG. 7. The recombinant heparinase III (●), the H295Amutant enzyme (∘), and the H510A mutant enzyme () were concentrated andbuffer exchanged into 50 mM sodium phosphate buffer, pH 7.0. Readingswere taken using a quartz cell with a 1 mm path length at 25° C. Spectrawere recorded between 205 and 260 mn with an average of 10 scans; thebandwidth was 1.0 nm; and the scan rate was 3 m/min. The CD bandintensities are expressed as molar ellipticities, θ_(M), indegrees·cm²·dmol⁻¹.

The possibility remained that the histidine 295 and/or histidine 510were somehow responsible for the folding or the tertiary structure ofthe enzyme and not directly involved in catalysis. However, the CDspectrum for H295A and H510A were nearly identical to that ofrecombinant heparinase III (FIG. 7). While the near identity of the CDprofiles does not preclude the possibility that there are perturbationsin the local environment surrounding histidine 295 and histidine 510that are not represented in the CD profile, it does suggest there are nogross conformational changes induced by mutating histidine 295 andhistidine 510 to alanine.

Example 4

Comparison of the Heparinase I and III re Tumor Growth and Metastasis

Heparinases I and III, which have very distinct substrate specificitiesfor cleaving HLGAGs, were employed as tools to investigate the role ofHLGAGs in tumor growth and metastasis. While heparinase I cleaves at thehighly sulfated regions of HLGAGs, heparinase III only cleaves at theunder-sulfated regions of the polysaccharide chain, thereby renderingthese enzymes powerful tools to investigate in vivo and in vitro rolesof HLGAGs, in development, morphogenesis, angiogenesis etc. To examinethe roles of HLGAGs in tumor growth and metastasis, we used B16BL6melanoma as a model system and treated tumor-bearing mice with eitherheparinases I or III to investigate both primary tumor growth as well astumor metastasis. Consistent with the current paradigm, heparinase Iaccelerated tumor growth (FIG. 8). At a dosage of 0.5 mg/kg/day ofheparinase I, tumor growth was increased by about 39%. However and mostsurprisingly, heparinase III inhibited primary tumor growth (FIG. 8).The inhibition of melanoma growth by heparinase III was shown to be dosedependent. Inhibition of primary tumor growth by heparinase III wasfirst observed at 2 mg/kg per day. Tumor growth was inhibited by 73% at12 mg/kg per day, the maximum dosage tested in the study (FIG. 8).Control mice treated with heat inactivated heparinase III exhibitedcomparable growth curves with that of mice treated with PBS (FIG. 8),suggesting that the catalytic activity of heparinase III was responsiblefor heparinase III's ability to inhibit primary tumor growth.Histological examination of tumor samples revealed increased apoptosisin heparinase III treated tumors, while heparinase I treated tumorsrevealed reduced apoptosis.

Mice, 15 days after tumor implantation with B16BL6 melanoma, wereexamined. 4×10⁵ log growth phase B16BL6 melanoma cells in 0.1 ml PBSwere injected to the flank of C57BL/6 mice on day 1. Daily injection of0.1 ml of PBS, heat inactivated hep III or active heparinase III (2mg/ml, recombinantly expressed, purified, buffer exchanged with PBS, andconcentrated) started at the day 4 and continued throughout theexperiment. At day 7, osmotic pumps (100 μl capacity delivering at 0.5μl per hour) containing PBS or 3 mg/ml hep III were implantedsubcutaneously at a place remote from the tumor site. Mice weresacrificed at day 15. Upon visual inspection, the control the micetreated with PBS or inactive Hep III had significantly larger tumormasses than the mice treated with active Hep III.

FIG. 8 depicts the tumor volume of the tumors isolated from the micedescribed above. Tumor volume was measured daily after day 7 with acaliper and calculated with the formula [volume=0.52×(width)²×(length)].The data was depicted as growth curves of mice bearing melanoma treatedwith PBS, inactive hep III and active hep III.

To ensure that these observations were not limited to the tumor modelchosen, hep III was used to treat mice bearing Lewis lung carcinoma(LLC) tumors. Growth curves of primary tumor growth for LLC tumor inC57BL/6 mice treated with either PBS or heparinase III were plotted.4×10⁵ log phase LLC cells were injected subcutaneously to the flank ofmice on day 1. Daily injection of 0.1 ml of either PBS or 2 mg/mlrecombinantly expressed heparinase III started at the day 4 andcontinued throughout the experiment. At day 8, osmotic pumps (100 μlcapacity delivering at 0.5 μl per hour) containing PBS or 3 mg/ml hepIII were implanted subcutaneously at a place remote from the tumor site.Mice were sacrificed at day 20. Lung metastasis of LLC cells injectedthrough tail vein were quantitated as number of lung nodules. Log growthLLC cells were trypsinized for 30 seconds and resuspended in PBS to afinal concentration of 1×10⁶ per ml. For experimental group, cells wereincubated with 200 nm hep III for 30 minutes at 37° C. before injecting0.2 ml of cell suspension via tail vein. Mice were euthanized 12 daysafter tail vein injections, lungs were harvested, rinsed in tap waterand fixed overnight in Bouin's solution. The number of nodules on lungsurface was counted with the aid of a dissection microscope.

Similar to the B16BL6, heparinase III treatment of mice-bearing LLCtumor at 12 mg/kg per day showed inhibition in tumor growth. Inaddition, removal of the HLGAG coat present on the LLC cells uponheparinase III treatment (and the presence of HLGAG fragments) inhibitedthe LLC cells to colonize in the lungs similar to the B16BL6 experiment.We investigated the ability of HLGAG fragments derived from LLC cells toeither support or inhibit B16BL6 tumor growth and metastasis. Both,heparinase I and III generated HLGAG fragments from the LLC cells wereisolated, harvested in PBS. Consistent with the B16BL6 results,heparinase I generated LLC HLGAG fragments promoted growth of B16BL6tumor cells, while heparinase III generated LLC HLGAG fragments showedminimal effect on growth of B16BL6 cells. Similarly, when B16BL6 cellswere incubated with the heparinase derived LLC HLGAG fragments prior toinjection into mice, heparinase III derived LLC HLGAG fragmentsinhibited B16BL6 metastasis to the lungs. Thus, the in vivo studies,along with in vitro cell culture experiments points to the enzymaticaction of hep III reducing the tumorigenicity of a variety of tumor celltypes.

There are two possible mechanisms by which heparinase may be acting ontumor cells. For instance, heparinase III treatment of cells may resultin cells losing their unique surface HLGAG coat and this directly orindirectly may impinge on their ability to grow or metastasize. On theother hand heparinase III treatment of cells may also result in thegeneration of distinct HLGAG fragments, and these fragments could thendirectly or indirectly modulate tumor cell function. It was thought thatheparinase III may function though either one of these mechanisms orthrough some combination of these mechanisms. To investigate themechanisms of action further, we treated B16BL6 cells with eitherheparinases I or III, to remove the HLGAG coat on the tumor cellsurface. Interestingly, the removal of the HLGAG coat, either byheparinase I or III, had no effect on the ability of these cells to growin mice compared to untreated cells. As shown below, we also found thatHLGAG fragments were capable of modulating tumor cell function,suggesting that this is the mechanism through which heparinase IIIexerts its anti-tumor and anti-metastasis functions.

We next investigated the ability of heparinase treated B16BL6 melanomacells to metastasize to the lungs. Lung metastasis of B16BL6 melanoma 13days after tail vein injection of B16BL6 cells is shown in FIG. 9.B16BL6 cells in log growth phase were briefly trypsinized andresuspended in PBS to a final concentration of 1×10⁶ per ml. Prior toinjection, cells were treated with either PBS, hep I (200 mn) or hep III(200 nm) for 30 min. at 37° C. 0.2 ml cell suspensions (2×10⁵ cells)were injected slowly via tail vein. Mice were sacrificed 13 days laterand lungs were harvested, rinsed with tap water. The number of noduleson lung surface were counted with the assistance of a dissectionmicroscope. *indicates p<0.05 (Mann-Whitney test).

B16BL6 cells were treated with either heparinases I or III and theninjected via tail vein of syngeneic mice. Interestingly, heparinase IIItreated B16BL6 cells were significantly less able to metastasize to thelungs, while heparinase I treated cells were marginally effected, ifany, in their ability to metastasize to the lungs when compared to thecontrol PBS treated cells (FIG. 9). Thus, the removal of specific HLGAGcoat present on the B16BL6 tumor cells significantly affected theability of the tumor cells to metastasize but had no effect on thegrowth of B16BL6 tumor cells. It should be pointed out that heparinasetreatment of cells would generate HLGAG fragments that might still bindto specific proteins on the B16BL6 surface to inhibit tumor metastasis.

In order to investigate a plausible role in tumor growth and metastasisfor the HLGAG fragments generated upon heparinase treatment of theB16BL6 HLGAG coat, both the heparinase I and III generated HLGAGfragments from the B16BL6 cells were isolated, harvested in PBS andtested (FIG. 10). B16BL6 melanoma were treated with GAG fragmentsgenerated from treatment of B16BL6 cells with hep I and III. Briefly,80-90% confluent cells were washed with PBS once. 1.5 ml of PBScontaining 200 nm of heparinase I or III were added to the flasks,incubated at 37° C. on a shaker for 2 h. Supernatant was pooled into atube, centrifuged for 5 minutes at 3000 rpm, boiled for 15 minutes andfiltered. The solution was finally incubated with Chondroitinase ABC for2 h at 37° C., the reaction was stopped by boiling for 1 min. 4×10⁵B16BL6 cells were injected subcutaneously as described in FIG. 8 onday 1. Osmotic pumps (200 ul capacity delivering 0.5 ul per hour) wereimplanted subcutaneously on day 2. Daily injection of 0.1 ml GAGfragment solution and PBS was started on day 5 and continued throughoutthe experiment. The mice were euthanized on day 15. Tumor volume isshown in FIG. 10A. Lung metastasis of B16BL6 melanoma were examined.2×10⁵ of B16BL6 resuspended in PBS, heparinase I generated fragment andheparinase III generated fragment solutions were injected via tail veinof mice (n=7 or 8). Lungs were harvested 13 days after injection,treated and counted as described earlier. * indicates p<0.05(Mann-Whitney test). The number of lung nodules was calculated.

Interestingly, heparinase I generated HLGAG fragments significantlypromoted primary tumor growth, while heparinase III generated fragmentsshowed did not (FIG. 10). Consistent with the enhanced tumor growth,histological examination of tumor samples revealed reduced apoptosis forheparinase I generated HLGAG fragment treatment. On the other hand andmost intriguingly, when B16BL6 cells were suspended in the PBScontaining heparinase III generated HLGAG fragments prior to injectionvia tail vein of mice, these fragments inhibited lung metastasis ofB16BL6 cells, while heparinase I generated fragments showed marginaleffect, if any. Thus, the HLGAG fragments generated from B16BL6 cells byheparinase treatment also appear to play a role in tumor growth andmetastasis.

In further support of this conclusion ex vivo digestion of the HLGAGcoat present on tumor cells with either heparinase, followed bycentrifugation and resuspension in PBS to remove the enzyme and HLGAGfragments released from the cell surface, prior to in vivo injectionresults in the cells being functionally identical to controls. Thus thereleased tumor cell HLGAG fragments appear to play the key role inmodulating tumor growth and metastasis.

Immunohistochemistry was done as described (Parangi et al., 1996;O'Reilly et al., 1994) with minor modifications. Briefly, tumor tissueswere fixed in either 4% (vol/vol) formaldehyde overnight for vonWillebrand factor (vWF) staining and terminal deoxynucleotidetransferase (TdT) labeling or in Glyo-Fixx solution overnight for Ki-67nuclear antigen staining. Tissues were embedded in paraffin according tostandard histological procedures. For vWF staining, sections (5 μmthick) were incubated with 0.2 N HCl for 10 min. autoclaved in a Coplinjar immersed with Target retrieval Solution (Dako) for 15 min. andpermeabilized with 2 μg/ml proteinase K at 37° C. for 15 min. Sectionswere incubated with rabbit anti-human vWF antibody coupled withhorseradish peroxidase (HRP) (Dako). Positive staining was detected bysubstrate reaction with diaminobenzidine. Sections were counterstainedwith Gill's hematoxylin and mounted in Permount (Fisher). Ki-67 antigenstaining (rabbit anti-human Ki-67 antigen antibody coupled with HRP,Dako) and TdT labeling (DeadEnd Colorimetric Apoptosis Detection System,Promega) were done essentially according to manufacture's protocol.Capillary density was determined by counting the number of vWF-positivecapillaries per high power field (HPF, ×200). The proliferative andapoptotic indices of tumor cells within areas of viable tumor wereestimated from the percentage of cells scored under a light microscopeat 400-fold magnification. A minimum of 2000 cells were counted in eachanimal. # indicates standard error.

The overall similarity of data for the B16BL6 and the LLC animal modelssuggests an important role for HLGAGs in tumor growth and metastasis.The differential effects of heparinases I and III, and the HLGAGfragments generated by heparinases are consistent with the uniquespecificities of heparinases, and hence the distinct oligosaccharideproducts they generate. Moreover, HLGAG fragments for one cell type isable to influence effects on another cell type, strongly suggesting theinvolvement of specific sequences of HLGAG in modulating effects ontumor growth and metastasis.

B16BL6 melanoma cells were transfected with antisense 2OST in pcDNA3.1and tested in an in vitro invasion assay. The cells that migrated wereremoved and counted. The number of cells migrated per high power field(×400) for antisense 2OST transfected cells was twice as much as that ofuntransfected B16BL6 cells. The results are shown in the bar graph ofFIG. 11.

The ability of the transfected cells to develop into primary tumors wasassessed by subcutaneous inoculation of 4×10⁵ B16BL6, transfected anduntransfected respectively into the flank of nude mice. The mean tumorvolume and tumor weight of transfected group was more than two foldgreater than that of the untransfected control group, as shown in FIG.12 a and b respectively.

The ability of the transfected cells to metastasize was determined byinjection of 2×10⁵ B16BL6 in 0.2 ml PBS, of transfected anduntransfected cells via tail vein of C57BL6 mice. The number ofmetastatic nodules on the lung surface for the transfected group wasmore than three fold greater than that of the untransfected control.Thus the 2OST antisense transfected B16BL6 appear to be more invasivewith higher metastatic potential and growth rate.

Example 5

HLGAG Fragments with Distinct Composition are Potent Inhibitors of TumorGrowth and Metastasis

Methods: B16BL6 cells were treated with either a, hep I; b, hep III; orc, a PBS control and the released HLGAG fragments harvested. Saccharidefragments were collected in PBS, and subjected to purification andfractionation. First, samples were bound to an Ultrafree-DEAE membrane,which had been equilibrated with pH 6.0 sodium phosphate, 0.15 M NaCl.They were washed with the same buffer and eluted with 0.1 M sodiumphosphate buffer pH 6.0 that contained 1.0 M NaCl. The fragments werethen concentrated and buffer exchanged into ultrapure water byapplication to a microcon column (MWCO=3,000 Da). The sample wasdigested overnight with a cocktail of hep I-III (1 mU each) in 25 mMsodium acetate 1 mM calcium acetate, pH 7.0. Analysis was completed bycapillary electrophoresis using a high sensitivity flow cell underreverse polarity with a running buffer of 50 mM tris/phosphate pH 2.5.Disaccharide identification was made by comigration with knownstandards, identity of peaks is enumerated in a and b. d, Table showingthe relative percentage of the HLGAG disaccharides in hep I and hepIII-generated fragments. The percentage was obtained based on thenormalized peak areas of the different disaccharides in a and b. Notethat the relative composition of the hep I and hep III-generatedfragments are very different. The alphanumeric assignment of eachdisaccharide is also listed as outlined previously (Venkataraman, G.,Shriver, Z., Raman, R. & Sasisekharan, R. Sequencing complexpolysaccharides. Science 286, 537-42 (1999).). Saccharide analysis ofthe B16BL6 cells that were transfected with the 2OST(−) indicated thatthere was an absence of 2-O sulfate-containing saccharides, specificallythe trisulfated disaccharide ΔU₂₅-H_(NS,6S). Mass spectrometricoligosaccharide mapping of hep I (e) and hep III (f) derived HLGAGsaccharide fragments. Hep I or hep III-derived HLGAG saccharidefragments were subjected to partial enzymatic cleavage by 100 mM (8μg/ml) heparinase II in 10 mM ethylenediamine, 10 μM ovalbumin, 1 μMdextran sulfate pH 7.0 for one hour. Resulting digests were complexedwith the basic peptide (RG)₁₉R and subjected to matrix-assisted laserdesorption ionization mass spectrometry. The HLGAG fragment fingerprintis different for the hep I vs hep III generated fragments consistentwith each being structurally distinct.

Results: Compositional studies of the HLGAG saccharide fragmentsgenerated upon heparinase treatment confirmed that the HLGAG fragmentsreleased from B16BL6 cells by hep I or hep III are compositionallydifferent and structurally distinct (FIG. 13 a-f). Capillaryelectrophoresis, in combination with exhaustive enzymatic digest, wasused to derive compositional information on the saccharide fragments(FIG. 13). The saccharide fragments derived from hep III treatment hadmore of tri and di-sulfated disaccharides while the hep I-treated HLGAGshad more mono and un-sulfated disaccharides (FIG. 13 a, b). This isconsistent with the known substrate specificities of the heparinases. Inaddition, mass spectrometric investigation of HLGAGs, yielded a“fingerprint” of oligosaccharides generated from each of the treatmentsand proved that the saccharide fragments generated from the differenttreatments are structurally distinct (FIG. 13 e, f).

The compositional analysis of the 2-OST antisense construct,demonstrating the deficiency that these mutants have in sulfated HLGAGsspecifically 2-O sulfation. Comparison between the composition of thecell surface HLGAGs for this mutant and those for hep I and hep IIIgenerated fragments from non-transfected B16BL6 cells indicates that theHLGAGs of the mutant are chemically closer in comparison to hep Igenerated fragments than of hep III generated fragments.

Example 6

Mechanism of Action: HLGAGs Impinge on the Biological Activity ofSpecific Signaling Molecules

Having observed the marked and opposite effects that distinct HLGAGfragments have on both the tumor and vascular compartments, we sought toelucidate the underlying molecular mechanism of HLGAGs in tumorprogression. As many HLGAG binding proteins are growth factors andcytokines, we therefore systematically explored HLGAG-binding growthfactors that play key roles in tumor pathobiology to identify animmediate target of the HLGAG fragments generated from the surface oftumor cells. FGF2 signaling has been shown to be a prerequisite formelanoma progression promoting tumor growth in an autocrine fashion, andthe interruption of the FGF2 autocrine loop by interfering with eitherFGF2 or FGF receptor (FGFR) activity results in inhibition of melanomaprogression (Rodeck, U. et al. Constitutive expression of multiplegrowth factor genes by melanoma cells but not normal melanocytes. JInvest Dermatol 97, 20-6 (1991). Becker, D., Meier, C. B. & Herlyn, M.Proliferation of human malignant melanomas is inhibited by antisenseoligodeoxynucleotides targeted against basic fibroblast growth factor.Embo J 8, 3685-91 (1989). Becker, D., Lee, P. L., Rodeck, U. & Herlyn,M. Inhibition of the fibroblast growth factor receptor 1 (FGFR-1) genein human melanocytes and malignant melanomas leads to inhibition ofproliferation and signs indicative of differentiation. Oncogene 7,2303-13 (1992). Torcia, M. et al. Interferon-alpha-induced inhibition ofB16 melanoma cell proliferation: interference with the bFGF autocrinegrowth circuit. Biochem Biophys Res Commun 262, 838-44 (1999).). On theother hand, upregulation of the expression of FGF2 in normal melanocytesresult in their malignant transformation (Nesbit, M. et al. Basicfibroblast growth factor induces a transformed phenotype in normal humanmelanocytes. Oncogene 18, 6469-76 (1999).). Furthermore, FGF2 is apotent and essential angiogenic factor regulating melanomaneovascularization (Wang, Y. & Becker, D. Antisense targeting of basicfibroblast growth factor and fibroblast growth factor receptor-1 inhuman melanomas blocks intratumoral angiogenesis and tumor growth. NatMed 3, 887-93 (1997). Birck, A., Kirkin, A. F., Zeuthen, J. &Hou-Jensen, K. Expression of basic fibroblast growth factor and vascularendothelial growth factor in primary and metastatic melanoma from thesame patients. Melanoma Res 9, 375-81 (1999).) Most importantly,specific HLGAG structures are known to bind and modulate FGF2 activity,and there is increasing evidence that, HLGAG sequences, depending ontheir structure, can either promote or inhibit FGF2 activity (Guimond,S. E. & Turnbull, J. E. Fibroblast growth factor receptor signaling isdictated by specific heparan sulphate saccharides. Curr Biol 9, 1343-6(1999).). Given the multiple lines of evidence implicating FGF2 as a keyswitch in melanoma progression, taken together with FGF's strictdependence on HLGAGs for its activity, we sought to determine whetherthe immediate target of tumor-derived HLGAG fragments is indeed FGF2.

To test whether hep I and hep III-derived fragments bind to FGF2 andaffect its activity, we first established that hep III treatment inhibitFGF-induced proliferation of B16BL6 cells in vitro, and additionally anddirectly confirmed this by examining FGF-mediated downstream signalingpathways, viz., the MAP kinase pathway (ie., Erk-1, 2), the principlesignal transduction pathway of FGF2 leading to cell proliferation anddifferentiation (Seger, R. & Krebs, E. G. The MAPK signaling cascade.Faseb J 9, 726-35 (1995).).

a. Methods: B16BL6 cells in 10 cm culture dishes were serum starved for48 hours before stimulation with 50 ng/ml FGF2. Cells were stimulatedfor 20 minutes before whole cell lysates were prepared with 1 mlmodified RIPA buffer containing various enz me inhibitors (50 mMTris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 EDTA,1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, leupeptin, and pepstatin, 1 mMactivated Na₃VO₄, 1 mM NaF). Protein concentration in the lysate wasdetermined using the Bio-Rad protein assay kit (BioRad) and adjustedaccordingly for electrophoresis analysis. For the heparinase treatedgroups, cells were treated with either hep I or hep III (200 nM) for 30mm at 37° C. prior to addition of FGF2. The immunoblot was probed withanti-Erk-1, 2 or anti-phospho-Erk-1, 2 antibody (New England Biolabs;MA) and detected by anti-rabbit IgG conjugated to HRP using SuperSignalWest Pico Chemiluminescent substrate (Pierce, Ill).

b. FGF-mediated proliferation of BaF3 cells with transfected FGFR in thepresence of HLGAG fragments generated with either hep I or III BaF3cells expressing FGFR were grown in the following fashion. The initialcell number was counted by Coulter counter, and resuspended to a densityof 1×10⁵ cells/ml into 12 samples of 6 ml. Each sample of cells wascentrifuged 3 min at room temperature at 1085×g, and resuspended inHLGAG preparations in PBS, producing two sets of cells in the samemedia. One of each set was supplemented with 50 ng/ml FGF2 () while theother was unsupplemented (). 1 ml from each set was added to each of 3wells on a 24-well tissue culture plate. The cells were incubated for 72hr at 37° C./5% CO₂. Whole cell number was counted at the experimentalendpoint by Coulter counter. This procedure was repeated three times.Collected data was normalized using a proliferative index (PI), aspreviously described (Padera, R., Venkataraman, G., Berry, D.,Godavarti, R. & Sasisekharan, R. FGF-2/fibroblast growth factorreceptor/heparin-like glycosaminoglycan interactions: a compensationmodel for FGF-2 signaling. Faseb J 13, 1677-87 (1999).). The index isdefined as the increase in cell number for the experimental case dividedby the increase in cell number for the positive control. The positivecontrol was cells in DMEM with 10% BCS, 2 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin, 500 ng/ml heparin, and 50 ng/mlFGF2. The negative control was cells in DMEM with 10% BCS, 2 mML-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 ng/mlheparin.

c, Effect of treatment of tumor with hep I and hep III in FGFR1activation compared to PBS. Level of phosphorylated FGFR1 in tumorsamples was assessed by standard immunoprecipitation followed byWesternblotting with phosphotyrosine specific antibody. Primary B16BL6tumors were grown and treated as described earlier and at day 15 thetumor was harvested in cold modified RIPA buffer containing enzymeinhibitors and homogenized. The homogenates were past through 25-gaugeneedle 3 times and centrifuged. The supernatant was adjusted for proteinconcentration using the Bio-Rad protein assay kit (Bio-Rad). FGFR1 wasimmunoprecipitated with poly-clonal anti-FGFR1 antibody (Santa CruzBiotechnology, Inc., CA.). Samples were then pelleted, washed, andeluted from the beads by addition of sample buffer and boiled for 3minutes (Kapila, Y. L., Wang, S. & Johnson, P. W. Mutations in theheparin binding domain of fibronectin in cooperation with the V regioninduce decreases in pp125(FAK) levels plus proteoglycan-mediatedapoptosis via caspases. J Biol Chem 274, 30906-13 (1999).). Afterelectrophoresis, the gel was transferred to nitrocellulose membrane bystandard methods. The immunoblot was probed with phosphotyrosinespecific antibody conjugated to HRP (RC20; Transduction Laboratories,Lexington, Ky.) and developed with SuperSignal West PicoChemiluminescent substrate. The molecular weight of FGFR1 is 120 KDa.

d, Effect of heparinase treatment on activation of FAK in B16BL6 tumor.The FAK protein was immunoprecipitated with mouse anti-FAK monoclonalantibody (Transduction Laboratories, Lexington, Ky.) according to theprocedures described above. The phosphorylated FAK was detected usingphosphotyrosine specific antibody RC20.

e, Level of total and phosphorylated Erk-1, 2 in heparinase-treatedB16BL6 tumor. Tumor homogenates were prepared and processed as describedin c. The supernatant was used for total protein concentration assay andimmunoblotting. The immunoblot was detected as described in a.

f, Effect of heparinase treatment on Akt activation. The primary tumorwas treated and processed as described above. Akt antibody andphospho-Akt antibody from New England Biolabs were used to probe theimmunoblot.

Results: Upon FGF2 stimulation, decreased Erk-1, 2 activation was seenin hep III treated cells while increased Erk-1, 2 activation was seenwith cells treated with hep I. The in vitro data was further confirmedusing F32 cells, a pre-lymphocyte cell line that has been transfectedwith FGFR, and that often has been used as a model system to studyFGF-mediated signaling in cell culture unfettered by complicationsassociated with signaling events initiated by other growth factorsand/or receptors (Ornitz, D. M. et al. Receptor specificity of thefibroblast growth factor family. J Biol Chem 271, 15292-7 (1996).).Similar to what was observed in B16BL6 cells, hep I fragments promote,whereas hep III fragments inhibited FGF2-mediated cellular proliferationin these cells (FIG. 14). Together, the in vitro findings point to thefact that HLGAG fragments derived from the cell surface cansubstantially and specifically affect FGF2 signaling.

Consistent with the in vitro observations, we find that hep I and hepIII-derived B16BL6 fragments significantly affect FGF signaling pathwaysin vivo. Within the tumor in the animals, we examined both FGFRphosphorylation in hep I and hep III-treated animals as well as Erk-1and 2 signaling. Treatment of the primary tumor with hep III (or itsgenerated fragments) inhibited phosphorylation of FGFR1 while hep Itreatment had the opposite effect on the phosphorylation of FGFR1.Consistently, treatment of the primary tumor with hep III resulted in alower level of activated Erk-1, 2. Additional intracellular signalingevents such as focal adhesion kinase (FAK) activity, which is implicatedin cell adhesion and migration processes (Rodriguez-Fernandez, J. L. Whydo so many stimuli induce tyrosine phosphorylation of FAK? Bioessays 21,1069-75 (1999). Schlaepfer, D. D., Hauck, C. R. & Sieg, D. J. Signalingthrough focal adhesion kinase. Prog Biophys Mol Biol 71, 435-78(1999).), was similarly modulated by hep I and III treatment of thetumor. Consistent with these findings, hep III treatment inhibited FAKactivation. Notably there was no change in activation of Akt with eitherhep I or hep III treatment, indicating that the changes inphosphorylation were specific and resulted from down-regulation of onlycertain signaling pathways. Together, these results suggest that HLGAGfragments mediate FGF2 signaling with hep I-derived fragments promotingFGF2 activity and hep III-generated fragments inhibiting it. This effectwas observed in key steps of FGF-mediated signaling, from the cellsurface receptor (FGFR) through downstream signaling events.

Example 7

Modulation of FGF2 Activity in vivo by B16BL6Fragments

Methods: Assessment of FGF2 signaling in vivo with the rat cornealpocket assay. Representative slit lamp photographs of rat corneas on day6 after implantation with Hydron pellets containing FGF2, hep Ifragments with FGF2, and hep III fragments with FGF2. The amount of FGF2loaded into each pellet was ˜120 ng, and the amount of HLGAG fragmentswas approximately 1 ng. The pellets were prepared and implantedessentially as described (Kenyon, B. M. et al. A model of angiogenesisin the mouse cornea. Invest Ophthalmol Vis Sci 37, 1625-32 (1996)). Onday 6 after the implantation into the cornea of Sprague-Dawley rats(n=5), the corneal neovascularization was photographed with a slit lampand the extent of neovascularization was expressed as linear length andcircumferential clock hours as described (Kenyon, B. M. et al. A modelof angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci 37,1625-32 (1996)). Results are summarized in the table. Control pellets cotaming no FGF2 failed to induce neovascularization. It was noted thatthe inhibition of neovascularization by hep III derived fragments aredose dependent, with initial inhibition observed at about 0.02ng/pellet. In addition, the inhibition of neovascularization by hep IIIderived fragment was found to be independent of the site ofimplantation, with similar inhibition observed when hep III derivedfragments was implanted as a second pellet in between the FGF2 onlypellet d the limbus. Indicates mean and SE.

Model of the formation of cryptic HLGAG modulators of FGF2 signaling.Interaction of HLGAGs (as part of proteoglycans) with theheparin-binding domains of FGF2 and FGFR allows the formation of aternary complex at the cell surface that forms e basis of FGF2signaling. Digestion of the cell HLGAG coat with hep I releasesfragments with an appropriate spatial display of 2 O-, 6 O- andN-sulfated groups that would allow an optical “fit” to both FGF2 andFGFR, leading to signaling through tyrosine kinase activation.Conversely, hep III-generated HLGAG fragments display another pattern ofsulfated groups are still able to bind FGF2 but fail to form aconstructive signaling complex at the cell surface, thus inhibiting FGF2activity.

Results: To demonstrate a direct interaction between B16BL6 HLGAGfragments and their immediate target FGF2 in vivo, we evaluated theability of B16BL6 HLGAG fragments to modulate FGF2-induced responsesleading to cell migration, proliferation, and differentiation in vivousing corneal neovascularization assay (FIG. 15 a table). In this model,hep I-generated fragments mixed with FGF2 bound to the growth factor andpromoted the in vivo neovascularization response to FGF2 (FIG. 15 atable) whereas hep III-generated fragments, mixed with FGF2, bound tothe growth factor but dramatically inhibited its activity (FIG. 15 atable). This result is consistent with the changes in neovascularizationobserved in the immunohistological study of tumor. Taken together, theabove results indicate that a direct in vivo target of the HLGAGfragments released by heparinase treatment is FGF2. Thus, it can beconcluded that hep I-generated fragments act to bridge FGF2 to itscognate receptor activating intracellular signaling pathways, while hepIII-derived fragments are antagonists, preventing the formation of asignaling complex at the cell surface. Thus, by either directlyactivating or inhibiting FGF2 signaling, these bioactive fragments arepotent modulators of tumor growth and metastasis. The results presentedhere do not preclude a direct or indirect effect of HLGAG fragments onother HLGAG binding growth factors playing a role in tumorpathophysiology. However, based on the many lines of evidence presentedhere, it appears that FGF2 is indeed an immediate target forenzymatically derived HLGAG fragments.

The results presented herein demonstrate that by impinging on thebiological activity of specific signaling molecules, HLGAGs play adirect role in tumor growth and metastasis. Most importantly, HLGAGs atthe cell surface of tumor cells contain both ‘activatory’ and‘inhibitory’ HLGAG sequences that are in balance (FIG. 15 b). Thespecific degradation of one set of sequences (eg., by hep I) results inthe release of fragments that promote the biological activity ofHLGAG-binding signaling molecules, and thus act as a switch for tumorgrowth and metastasis. Conversely, degradation by an enzyme with anorthogonal substrate specificity (eg., hep III) tips the balance in theopposite direction, releasing fragments that antagonize HLGAG-bindingsignaling molecules, leading to the inhibition of tumor growth andmetastasis. Thus, we have demonstrated here for the first time thatchemically complex HLGAGs at the cell surface are “cryptic” promoters orinhibitors of tumor growth and metastasis that become biologicallyactive upon their release from the cell surface by specific HLGAGdegrading enzymes.

Just as collagenases clip the proteinaceous compartment of the ECM,serving either to increase tumor growth (eg., breakdown of the basementmembrane) or to inhibit tumors (eg., the formation of endostatin fromcollagen XVIII), the polysaccharide compartment exhibits a similarphenomenon. Importantly, like the proteolytically cleaved collagenfragment endostatin, distinct HLGAG oligosaccharides upon release byenzymatic cleavage from the tumor cell surface can serve as potentinhibitors of tumor progression. Thus, the present study not only allowsa new paradigm of how the polysaccharides modulate tumor growth andmetastasis, but it identifies a novel therapeutic target by providing aframework towards the development of HLGAG-based novel anti-cancermolecules.

The data presented herein demonstrate important findings relating to thepossible mechanisms and physiological implications of how HLGAGsregulate tumor growth and metastasis. HLGAG fragments may exert theireffects through many pathways including autocrine growth and angiogenicfactors, or through interactions with ECM molecules. Additionally, thesources of the endogenous HLGAG-degrading enzymes and it substratespecificity also become important. Production of HLGAG-degrading enzyme,presumably by the tumor cells, with substrate specificity similar toheparinase I will be advantageous to tumor cells. Secretion ofHLGAG-degrading enzyme by a tumor cell would lead to the production ofspecific HLGAG sequences (from it own coat or the tumor bed ECM) whichmight exert effects via autocrine and angiogenic growth factors, orthrough other signaling pathways to support tumor growth and metastasis.On the other hand production of an HLGAG-degrading enzyme with substratespecificity similar to heparinase III would be extremely beneficial tothe host. For instance, the endothelial cells in the vicinity of a tumoror macrophages can secrete an enzyme with substrate specificity ofheparinase III leading to the production of specific HLGAG sequencesthat inhibit tumor growth and metastasis. Consistent with such a modelpriming the animal with heparinase III does significantly inhibit tumorgrowth, suggesting a tumor-suppressor property of heparinase III. Abalance in the regulation of the bioavailibility of unique HLGAGsequences through HLGAG-degrading enzymes or through other mechanismsmay play a key switch to either support or inhibit tumor growth andmetastasis.

As described above, hep III treatment caused a significant inhibition inprimary tumor growth both in nude mice and C57BL/6 mice withsubcutaneous injections of hep III. These results indicate that theresponse to hep III treatment is not dependent on route ofadministration nor is it immune mediated.

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

1. A substantially pure heparinase III, comprising: a polypeptide havingthe amino acid sequence of the mature peptide of SEQ ID NO: 2, whereinat least one histidine residue selected from the group consisting of His36, His105, His110, His139, His152, His225, His234, His241, His424,His469, and His539 has been substituted with a residue selected from thegroup consisting of alanine, serine, tyrosine, threonine, and lysine. 2.A substantially pure heparinase III comprising: a modified heparinaseIII having a modified product profile, wherein the modified productprofile of the modified heparinase III is at least 10% different than anative product profile of a native heparinase III, wherein thesubstantially pure heparinase III has the amino acid sequence of themature peptide of SEQ ID NO: 2 where at least one histidine residueselected from the group consisting of His 36, His105, His110, His139,His152, His225, His234, His241, His424, His469, and His539 has beensubstituted with a residue selected from the group consisting ofalanine, serine, tyrosine, threonine, and lysine.
 3. A substantiallypure heparinase III comprising: a modified heparinase III that cancleave a heparan sulfate substrate having modified heparinase IIIk_(cat) value, wherein the modified heparinase III k_(cat) value is atleas 10% different than a native heparinase III k_(cat) value, whereinthe substantially pure heparinase III has the amino acid sequence of themature peptide of SEQ ID NO: 2 where at least one histidine residueselected from the group consisting of His 36, His105, His110, His139,His152, His225, His234, His241, His424, His469, and His539 has beensubstituted with a residue selected from the group consisting o alanine,serine, tyrosine, threonine, and lysine.
 4. A pharmaceutical preparationcomprising a sterile formulation of the substantially pure heparinaseIII of claim 1 and a pharmaceutically acceptable carrier.
 5. Animmobilized substantially pure modified heparinase III comprising: amodified heparinase III as in claim 1, and a solid support, wherein themodified heparinase III is immobilized on the solid support.
 6. Thesubstantially pure heparinase III of claim 1, wherein the polypeptidehas at least one substitution within a histidine residue selected fromthe group consisting of His 110, His225, and His241.
 7. Thesubstantially pure heparinase III of claim 1, wherein the polypeptidehas a substitution at His110.
 8. The substantially pure heparinase IIIof claim 1, wherein the polypeptide has a substitution at His241.
 9. Thesubstantially pure heparinase III of claim 1, wherein the polypeptidehas a substitution at His225.
 10. The substantially pure heparinase IIIof claim 9, wherein the His225 is substituted with alanine.